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1.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
2.
Plant Sci ; 310: 110961, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315586

RESUMO

Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the proportion of vacuolar Cd in protoplasts showed its ultimate storage capacity (82.24 %-83.40 %), and the Cd concentration stored in the protoplast maintained at a certain level (73.81-77.46 mg L-1). Besides, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2 = 0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.


Assuntos
Cádmio/toxicidade , Raízes de Plantas/metabolismo , Solanum nigrum/metabolismo , Antioxidantes/metabolismo , Glutationa/metabolismo , Microscopia de Fluorescência , Raízes de Plantas/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Solanum nigrum/efeitos dos fármacos
3.
Methods Mol Biol ; 2309: 201-218, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028689

RESUMO

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.


Assuntos
Arabidopsis/metabolismo , Bioensaio , Técnicas Biossensoriais , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Protoplastos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Medições Luminescentes , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais
4.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807467

RESUMO

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.


Assuntos
Citocininas/metabolismo , Oryza/genética , Protoplastos/metabolismo , Citocininas/imunologia , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Oryza/imunologia , Oryza/metabolismo , Reguladores de Crescimento de Plantas/genética , Proteínas de Plantas/genética , Brotos de Planta/genética , Regiões Promotoras Genéticas/genética , Protoplastos/imunologia , Transdução de Sinais/imunologia
5.
Plant Mol Biol ; 106(1-2): 145-156, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33694047

RESUMO

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.


Assuntos
Diterpenos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Linhagem Celular , Compostos de Epóxi/metabolismo , Proteínas de Membrana Transportadoras/química , Mutagênese/genética , Filogenia , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Estabilidade Proteica , Protoplastos/metabolismo , Tabaco/genética , Transcrição Genética , Tripterygium/genética
6.
Methods Mol Biol ; 2297: 105-113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33656674

RESUMO

Cryptochromes (CRYs) belong to an ancient and conserved class of blue-light receptor regulating circadian clock and development in animals and plants. Arabidopsis CRY2 form physiologically active homodimers in response to blue light treatment and further oligomerize into photobodies, which are expected to be the foci harboring protein interaction, phosphorylation, and ubiquitination. Here we describe two efficient methods developed to test the formation of blue-light-dependent photobodies of CRY-GFP fusing proteins using the mesophyll protoplasts of Arabidopsis or soybean, respectively.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Criptocromos/metabolismo , Protoplastos/metabolismo , Soja/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos , Criptocromos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Luz , Fosforilação , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismo , Soja/genética , Soja/metabolismo , Ubiquitinação
7.
Appl Biochem Biotechnol ; 193(8): 2455-2469, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33765267

RESUMO

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by two-dimensional analysis. Overexpression of XYL1, XYL2, XKS, and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH, and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.


Assuntos
Proteínas Fúngicas/sangue , Regulação Fúngica da Expressão Gênica , Protoplastos/metabolismo , Saccharomyces cerevisiae , Saccharomycetales , Xilose/metabolismo , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
8.
Nat Commun ; 12(1): 1007, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579946

RESUMO

Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5' maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.


Assuntos
Resistência à Doença/fisiologia , Ribonuclease P/genética , Ribonuclease P/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Vírus do Mosaico/genética , Vírus do Mosaico/metabolismo , Vírus de Plantas/genética , Protoplastos/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/genética , Ribonuclease P/química
9.
Nat Chem Biol ; 17(5): 549-557, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33633378

RESUMO

How aerobic organisms exploit inevitably generated but potentially dangerous reactive oxygen species (ROS) to benefit normal life is a fundamental biological question. Locally accumulated ROS have been reported to prime stem cell differentiation. However, the underlying molecular mechanism is unclear. Here, we reveal that developmentally produced H2O2 in plant shoot apical meristem (SAM) triggers reversible protein phase separation of TERMINATING FLOWER (TMF), a transcription factor that times flowering transition in the tomato by repressing pre-maturation of SAM. Cysteine residues within TMF sense cellular redox to form disulfide bonds that concatenate multiple TMF molecules and elevate the amount of intrinsically disordered regions to drive phase separation. Oxidation triggered phase separation enables TMF to bind and sequester the promoter of a floral identity gene ANANTHA to repress its expression. The reversible transcriptional condensation via redox-regulated phase separation endows aerobic organisms with the flexibility of gene control in dealing with developmental cues.


Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hidroponia/métodos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Oxirredução , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Espécies Reativas de Oxigênio/uso terapêutico , S-Adenosilmetionina/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transformação Genética
10.
Dev Cell ; 56(4): 557-568.e6, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33400914

RESUMO

Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.


Assuntos
Estudos de Associação Genética , Locos de Características Quantitativas/genética , Análise de Sequência de RNA , Análise de Célula Única , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Protoplastos/metabolismo , Reprodutibilidade dos Testes , Transcriptoma/genética
11.
Cells ; 10(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440743

RESUMO

Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are composed of several polysaccharides, of which cellulose forms the main fibrillar network. Enmeshed between these fibrils of cellulose are non-cellulosic polysaccharides (NCPs), pectins, and proteins. This study investigates the sequence, timing, patterning, and architecture of cell wall polysaccharide regeneration in suspension culture cells (SCC) of the grass species Lolium multiflorum (Lolium). Confocal, superresolution, and electron microscopies were used in combination with cytochemical labeling to investigate polysaccharide deposition in SCC after protoplasting. Cellulose was the first polysaccharide observed, followed shortly thereafter by (1,3;1,4)-ß-glucan, which is also known as mixed-linkage glucan (MLG), arabinoxylan (AX), and callose. Cellulose formed fibrils with AX and produced a filamentous-like network, whereas MLG formed punctate patches. Using colocalization analysis, cellulose and AX were shown to interact during early stages of wall generation, but this interaction reduced over time as the wall matured. AX and MLG interactions increased slightly over time, but cellulose and MLG were not seen to interact. Callose initially formed patches that were randomly positioned on the protoplast surface. There was no consistency in size or location over time. The architecture observed via superresolution microscopy showed similarities to the biophysical maps produced using atomic force microscopy and can give insight into the role of polysaccharides in PCWs.


Assuntos
Parede Celular/metabolismo , Celulose/metabolismo , Lolium/citologia , Regeneração , Xilanos/metabolismo , beta-Glucanas/metabolismo , Parede Celular/ultraestrutura , Células Cultivadas , Lolium/ultraestrutura , Protoplastos/metabolismo , Suspensões , Fatores de Tempo
12.
Nucleic Acids Res ; 49(2): 1133-1151, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406240

RESUMO

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.


Assuntos
Processamento Alternativo/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Silenciamento de Genes , Degradação do RNAm Mediada por Códon sem Sentido , Isoformas de Proteínas/genética , Interferência de RNA , Precursores de RNA/metabolismo , RNA de Plantas/metabolismo , Proteínas de Arabidopsis/biossíntese , Éxons , Genes de Plantas , Células HeLa , Humanos , MicroRNAs/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas/biossíntese , Protoplastos/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Fatores de Processamento de Serina-Arginina/biossíntese , Fatores de Processamento de Serina-Arginina/genética , Transcrição Genética , Transfecção
13.
Plant Sci ; 303: 110750, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33487339

RESUMO

Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Receptores de Superfície Celular/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Ácidos Indolacéticos/metabolismo , Microscopia Confocal , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismo
14.
Can J Microbiol ; 67(5): 406-414, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33226848

RESUMO

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.


Assuntos
Celulase/biossíntese , Hypocreales/enzimologia , Protoplastos/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Celulose/metabolismo , Regulação Fúngica da Expressão Gênica , Hypocrea/enzimologia , Hypocrea/genética , Hypocreales/genética , Microbiologia Industrial , Polimorfismo Genético , RNA Fúngico/genética , RNA Mensageiro/genética
15.
J Integr Plant Biol ; 63(3): 510-527, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33331695

RESUMO

Global warming poses a serious threat to crops. Calcium-dependent protein kinases (CDPKs)/CPKs play vital roles in plant stress responses, but their exact roles in plant thermotolerance remains elusive. Here, we explored the roles of heat-induced ZmCDPK7 in thermotolerance in maize. ZmCDPK7-overexpressing maize plants displayed higher thermotolerance, photosynthetic rates, and antioxidant enzyme activity but lower H2 O2 and malondialdehyde (MDA) contents than wild-type plants under heat stress. ZmCDPK7-knockdown plants displayed the opposite patterns. ZmCDPK7 is attached to the plasma membrane but can translocate to the cytosol under heat stress. ZmCDPK7 interacts with the small heat shock protein sHSP17.4, phosphorylates sHSP17.4 at Ser-44 and the respiratory burst oxidase homolog RBOHB at Ser-99, and upregulates their expression. Site-directed mutagenesis of sHSP17.4 to generate a Ser-44-Ala substitution reduced ZmCDPK7's enhancement of catalase activity but enhanced ZmCDPK7's suppression of MDA accumulation in heat-stressed maize protoplasts. sHSP17.4, ZmCDPK7, and RBOHB were less strongly upregulated in response to heat stress in the abscisic acid-deficient mutant vp5 versus the wild type. Pretreatment with an RBOH inhibitor suppressed sHSP17.4 and ZmCDPK7 expression. Therefore, abscisic acid-induced ZmCDPK7 functions both upstream and downstream of RBOH and participates in thermotolerance in maize by mediating the phosphorylation of sHSP17.4, which might be essential for its chaperone function.


Assuntos
Resposta ao Choque Térmico/fisiologia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Termotolerância/fisiologia , Zea mays/enzimologia , Zea mays/fisiologia , Ácido Abscísico/farmacologia , Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Peróxido de Hidrogênio/metabolismo , Mutação/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Termotolerância/efeitos dos fármacos , Termotolerância/genética , Zea mays/efeitos dos fármacos , Zea mays/genética
16.
Methods Mol Biol ; 2234: 87-98, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165782

RESUMO

In this protocol, we describe the establishment of a CRISPR/Cas9 system in Trichoderma reesei by generating a specific, codon-optimized Cas9-expressing strain and by in vitro transcription of a gRNA. This system induces mutagenesis or introduces a gene in a targeted way based on PEG-mediated protoplast transformation. Up to three targets, multiplexed genome editing can be obtained in one transformation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Fúngico , Hypocreales/genética , Sequência de Bases , Protoplastos/metabolismo , RNA Guia/genética , Transcrição Genética , Transformação Genética
17.
Methods Mol Biol ; 2200: 255-294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33175382

RESUMO

Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in molecular biology, vol 1062. Humana Press, Totowa, pp 509-537], describes the application of techniques of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing (a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei, and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are emphasized.


Assuntos
Arabidopsis/citologia , Citometria de Fluxo , Células Vegetais , Protoplastos/citologia , Arabidopsis/metabolismo , Protoplastos/metabolismo
18.
Methods Mol Biol ; 2170: 199-212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797460

RESUMO

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 µg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.


Assuntos
Fusarium/patogenicidade , Neurospora crassa/patogenicidade , DNA Complementar/genética , DNA Complementar/metabolismo , Fusarium/genética , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Neurospora crassa/genética , Protoplastos/metabolismo
19.
Anal Biochem ; 612: 114021, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33189704

RESUMO

We developed a binary vector co-expressing firefly luciferase (FF) and Renilla luciferase (REN) to detect protein stability in response to different stimuli, and verified the functionality of the vector. The StrigoQuant-like reporter expressing FF and REN in one transcript is a sensitive tool for detecting protein abundance in different genotypes. However, we found that significant differences in the relative FF/REN ratio of empty StrigoQuant vector in different genotypes. Therefore, to determine the actual protein abundance, the relative FF/REN ratio of the protein of interest should be normalized to that of the empty vector.


Assuntos
Bioensaio/métodos , Genes Reporter , Luciferases/metabolismo , Estabilidade Proteica , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vetores Genéticos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Indicadores e Reagentes/química , Lactonas/farmacologia , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Oryza/metabolismo , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo
20.
Nat Commun ; 11(1): 6191, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273474

RESUMO

In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulates CTI1 expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, "envelope docking" of ACCase permits fine-tuning of fatty acid supply during the plant life cycle.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos/biossíntese , Membranas Intracelulares/metabolismo , Acetatos/metabolismo , Acetil-CoA Carboxilase/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Radioisótopos de Carbono , Regulação da Expressão Gênica no Desenvolvimento , Luz , Simulação de Acoplamento Molecular , Plastídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Protoplastos/metabolismo
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