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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2836-2844, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472301

RESUMO

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.


Assuntos
Biologia Computacional , Tabaco , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Simulação por Computador , DNA Complementar , Filogenia , Melhoramento Vegetal , Tabaco/genética
2.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2845-2855, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472302

RESUMO

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.


Assuntos
Amido , Tabaco , Biomassa , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445105

RESUMO

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.


Assuntos
Lycopersicon esculentum/genética , Osmose/fisiologia , Pressão Osmótica/fisiologia , Solanum tuberosum/genética , Tabaco/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Transgenes/genética
4.
Int J Mol Sci ; 22(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34445241

RESUMO

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca2+) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species.


Assuntos
Temperatura Alta , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Termotolerância , Tabaco/metabolismo , Proteínas de Plantas/genética , Tubo Polínico/genética , Tabaco/genética
5.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445456

RESUMO

Flavonoids are representative secondary metabolites with different metabolic functions in plants. Previous study found that ectopic expression of EsMYB90 from Eutremasalsugineum could strongly increase anthocyanin content in transgenic tobacco via regulating the expression of anthocyanin biosynthesis genes. In the present research, metabolome analysis showed that there existed 130 significantly differential metabolites, of which 23 metabolites enhanced more than 1000 times in EsMYB90 transgenic tobacco leaves relative to the control, and the top 10 of the increased metabolites included caffeic acid, cyanidin O-syringic acid, myricetin and naringin. A total of 50 markedly differential flavonoids including flavones (14), flavonols (13), flavone C-glycosides (9), flavanones (7), catechin derivatives (5), anthocyanins (1) and isoflavone (1) were identified, of which 46 metabolites were at a significantly enhanced level. Integrated analysis of metabolome and transcriptome revealed that ectopic expression of EsMYB90 in transgenic tobacco leaves is highly associated with the prominent up-regulation of 16 flavonoid metabolites and the corresponding 42 flavonoid biosynthesis structure genes in phenylpropanoid/flavonoid pathways. Dual luciferase assay documented that EsMYB90 strongly activated the transcription of NtANS and NtDFR genes via improving their promoter activity in transiently expressed tobacco leaves, suggesting that EsMYB90 functions as a key regulator on anthocyanin and flavonoid biosynthesis. Taken together, the crucial regulatory role of EsMYB90 on enhancing many flavonoid metabolite levels is clearly demonstrated via modulating flavonoid biosynthesis gene expression in the leaves of transgenic tobacco, which extends our understanding of the regulating mechanism of MYB transcription factor in the phenylpropanoid/flavonoid pathways and provides a new clue and tool for further investigation and genetic engineering of flavonoid metabolism in plants.


Assuntos
Antocianinas , Brassicaceae/metabolismo , Perfilação da Expressão Gênica , Metabolômica , Proteínas de Plantas , Plantas Geneticamente Modificadas , Tabaco , Antocianinas/biossíntese , Antocianinas/genética , Brassicaceae/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
6.
Viruses ; 13(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34452461

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plantas Geneticamente Modificadas , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Estabilidade Proteica , Receptores de Coronavírus/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tabaco/genética , Tabaco/metabolismo , Células Vero
7.
Planta ; 254(3): 60, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34448043

RESUMO

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.


Assuntos
Inativação Gênica , RNA de Cadeia Dupla , Interferência de RNA , RNA Interferente Pequeno/genética , Tabaco/genética
8.
BMC Plant Biol ; 21(1): 359, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34353289

RESUMO

BACKGROUND: Plant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2. RESULTS: The inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2, 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2, which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture. CONCLUSIONS: A dominant dwarfing gene, BnaC04.BIL1, encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.


Assuntos
Brassica napus/crescimento & desenvolvimento , Brassica napus/genética , Proteínas de Plantas/genética , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Quinase 3 da Glicogênio Sintase/genética , Mutação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tabaco/genética
9.
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
10.
Commun Biol ; 4(1): 947, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373580

RESUMO

Land plant genomes carry tens to hundreds of Resistance (R) genes to combat pathogens. The induction of antiviral R-gene-mediated resistance often results in a hypersensitive response (HR), which is characterized by virus containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. Alternatively, systemic HR (SHR) is sometimes observed in certain R gene-virus combinations, such that the virus systemically infects the plant and PCD induction follows the spread of infection, resulting in systemic plant death. SHR has been suggested to be the result of inefficient resistance induction; however, no quantitative comparison has been performed to support this hypothesis. In this study, we report that the average number of viral genomes that establish cell infection decreased by 28.7% and 12.7% upon HR induction by wild-type cucumber mosaic virus and SHR induction by a single-amino acid variant, respectively. These results suggest that a small decrease in the level of resistance induction can change an HR to an SHR. Although SHR appears to be a failure of resistance at the individual level, our simulations imply that suicidal individual death in SHR may function as an antiviral mechanism at the population level, by protecting neighboring uninfected kin plants.


Assuntos
Cucumovirus/fisiologia , Regulação da Expressão Gênica de Plantas , Genes vpr/fisiologia , Doenças das Plantas/genética , Tabaco/virologia , Cucumovirus/genética , Resistência à Doença/genética , Proteínas de Plantas/genética , Tabaco/genética
11.
Plant Sci ; 310: 110971, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315589

RESUMO

The flowering-time gene FD encodes a bZIP transcription factor that interacts with FLOWERING LOCUS T (FT) to induce flowering in Arabidopsis. Previous research has identified two FT homologs of Platanus acerifolia, PaFT and PaFTL, which each have different expression patterns and are involved in diverse developmental processes. However, it is not known whether such FT/FD complexes participate in the flowering processes in P. acerifolia. Therefore, we isolated two closely related FD homologs, PaFDL1 and PaFDL2, and investigated their functions through the analysis of expression profiles, transgenic phenotypes, their interactions with different FT proteins, and potential cis-regulatory elements in their promoters. The PaFDL genes were found to display their maximal expression levels during the stage of floral transition, and subsequent expression patterns were also seen to be related to inflorescence developmental stage. In addition, both PaFDL1 and PaFDL2 were found to be subject to post-transcriptional alternative splicing, each gene producing two transcript forms. Transgenic tobacco overexpressing each of the four resulting transcript types displayed accelerated floral initiation and produced abnormal flowers. The results suggested that the complete PaFDL proteins may interact with different PaFT/PaFTL proteins in order to fulfill both conservative and diverse functions in floral initiation and floral development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flores/metabolismo , Flores/fisiologia , Proteínas de Plantas/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tabaco/genética , Tabaco/metabolismo
12.
Methods Mol Biol ; 2328: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251628

RESUMO

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas Repressoras/metabolismo , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Primers do DNA , Genes myc/genética , Luciferases/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Proteínas Repressoras/genética , Tabaco/genética
13.
Methods Mol Biol ; 2328: 253-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251631

RESUMO

Enhancers are one of the main classes of cis-regulatory elements (CREs) in the regulation of plant gene expression. Plant enhancers can be predicted based on genomic signatures associated with open chromatin. However, predicted enhancers need to be validated experimentally. We developed an experimental system for rapid enhancer validation. Predicted enhancer candidates are cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene. The construct is then agroinfiltrated into Nicotiana benthamiana leaves followed by bioluminescence signal detection and analysis. Positive bioluminescence signals indicate the enhancer function of each candidate, and the relative signal strength from different enhancers can be quantitatively measured and compared. In summary, we have developed an efficient and rapid plant enhancer validation assay based on a bioluminescent luciferase reporter and agroinfiltration-based N. benthamiana leaf transient expression. This assay can be used for the initial screening of candidate enhancers that are active in leaf tissue. The system can potentially be used to examine the activity of candidate enhancers under different environmental conditions.


Assuntos
Elementos Facilitadores Genéticos , Genes Reporter , Medições Luminescentes/métodos , Tabaco/metabolismo , Agrobacterium/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Vetores Genéticos , Luciferases/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Imagem com Lapso de Tempo , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
14.
Methods Mol Biol ; 2288: 293-305, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270019

RESUMO

Haploids are plants with gametophytic chromosome number, which upon chromosome duplication results in production of doubled haploids (DHs). There are several methods to obtain haploids and DHs, of which in vitro anther culture is the most effective and widely used method in tobacco. The production of haploids and DHs through androgenesis allows for a single-step development of complete homozygous lines from heterozygous genotypes, shortening the time required to produce homozygous genotypes in comparison to the conventional breeding scheme. The DH development process comprises two main steps: induction of androgenesis and duplication of the haploid genome. The critical stages of DH protocol in tobacco are determining the bud stage for anther culture, pretreatment, anther culture media, detection and identification of haploids, and chromosome doubling. Here we present an efficient anther culture protocol to get haploids and DHs in flue-cured virginia (FCV) tobacco. This optimized protocol can be used as a potential tool for generation of haploids and DHs for genetic improvement of tobacco.


Assuntos
Flores/crescimento & desenvolvimento , Melhoramento Vegetal/métodos , Técnicas de Embriogênese Somática de Plantas/métodos , Tabaco/crescimento & desenvolvimento , Meios de Cultura , Flores/genética , Haploidia , Pólen/genética , Pólen/crescimento & desenvolvimento , Tabaco/genética
15.
J Plant Physiol ; 263: 153464, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34225177

RESUMO

The environmental spread of hydrophobic pollutants has been receiving attention because of specific characteristics of these compounds that make them resistant to degradation, thus causing various toxic effects on humans as a result of their bioaccumulation. Here, we report the role of zucchini major-latex like proteins (MLPs) on the accumulation of hydrophobic pollutants, as consumption of contaminated crops is one of the main routes for accumulation. Transgenic tobacco plants expressing an aryl hydrocarbon receptor (AhR) gene with a ß-glucuronidase (GUS) inducible expression system were transformed with one of the three zucchini MLP genes (PG1, GR1, and GR3). MLP transgenic plants showed a significant increase in the fold induction of GUS activity compared to the parental AhR tobacco plants when one of the most toxic polychlorinated biphenyl (PCB) congeners, 3,3',4,4',5-pentachlorobiphenyl (CB126), was applied. GUS activity was detected in both aerial parts and roots after treatment with the strong carcinogen 3-methylcholanthrene. Phenotypic changes in the MLP tobacco during incubation with CB126 were also observed. The MLP transgenic plant PG1 responded to treatment with 0.32 nM CB126, whereas vector control plants significantly induced GUS activity at 200 nM CB126. Moreover, GUS activities in the MLP plants treated with other PCB congeners were significantly higher than those in the plants given the mock treatment. As GUS activities in the aerial parts of the plants were significantly correlated with the accumulation level of PCBs, these results strongly suggest that zucchini MLPs are related to the translocation of hydrophobic pollutants from the roots to the aerial parts through their binding affinity.


Assuntos
Transporte Biológico/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Raízes de Plantas/fisiologia , Bifenilos Policlorados/metabolismo , Poluentes do Solo , Tabaco/genética , Tabaco/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Plantas Geneticamente Modificadas
16.
Plant Cell Rep ; 40(9): 1735-1749, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34308490

RESUMO

KEY MESSAGE: Overexpression of the naturally occurring intron-retained (IR) forms of radish RsMYB1 and RsTT8 transcripts in Arabidopsis causes a substantial increase in anthocyanin accumulation. The production of anthocyanins in plants is tightly controlled by the MYB-bHLH-WD40 (MBW) complex. In this study, analysis of four radish (Raphanus sativus L.) inbred lines with different colored taproots revealed that regulatory genes of anthocyanin biosynthesis, RsMYB1 and RsTT8, produce three transcripts, one completely spliced and two intron retention (IR1 and IR2) forms. Transcripts RsMYB1-IR1 and RsMYB1-IR2 retained the 1st (380 nt) and 2nd (149 nt) introns, respectively; RsTT8-IR1 retained the 4th intron (113 nt); RsTT8-IR2 retained both the 3rd (128 nt) and 4th introns. Levels of most IR forms were substantially low in radish samples, but the RsTT8-IR2 level was higher than RsTT8 in red skin/red flesh (RsRf) root. Since all IR forms contained a stop codon within the intron, they were predicted to encode truncated proteins with defective interaction domains, resulting in the inability to form the MBW complex in vivo. However, tobacco leaves transiently co-expressing RsMYB1-IRs and RsTT8-IRs showed substantially higher anthocyanin accumulation than those co-expressing their spliced forms. Consistently, co-expression of constructs encoding truncated proteins with spliced or IR forms of their interaction partner in tobacco leaves did not result in anthocyanin accumulation. Compared with RsMYB1, the overexpression of RsMYB1-IRs in Arabidopsis pap1 mutant increased anthocyanin accumulation by > sevenfold and upregulated the expression of Arabidopsis flavonoid biosynthesis genes including AtTT8. Our results suggest that the stable co-expression of RsMYB1-IRs in fruit trees and vegetable crops could be used to increase their anthocyanin contents.


Assuntos
Antocianinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Raphanus/genética , Processamento Alternativo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Íntrons , Pigmentação/genética , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Tabaco/genética
17.
BMC Plant Biol ; 21(1): 299, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34187357

RESUMO

BACKGROUND: Cigar wrapper leaves are the most important raw material of cigars. Studying the genomic information of cigar tobacco is conducive to improving cigar quality from the perspective of genetic breeding. However, no reference genome or full-length transcripts at the genome-wide scale have been reported for cigar tobacco. In particular, anion channels/transporters are of high interest for their potential application in regulating the chloride content of cigar tobacco growing on coastal lands, which usually results in relatively high Cl- accumulation, which is unfavorable. Here, the PacBio platform and NGS technology were combined to generate a full-length transcriptome of cigar tobacco used for cigar wrappers. RESULTS: High-quality RNA isolated from the roots, leaves and stems of cigar tobacco were subjected to both the PacBio platform and NGS. From PacBio, a total of 11,652,432 subreads (19-Gb) were generated, with an average read length of 1,608 bp. After corrections were performed in conjunction with the NGS reads, we ultimately identified 1,695,064 open reading frames including 21,486 full-length ORFs and 7,342 genes encoding transcription factors from 55 TF families, together with 2,230 genes encoding long non-coding RNAs. Members of gene families related to anion channels/transporters, including members of the SLAC and CLC families, were identified and characterized. CONCLUSIONS: The full-length transcriptome of cigar tobacco was obtained, annotated, and analyzed, providing a valuable genetic resource for future studies in cigar tobacco.


Assuntos
Proteínas de Transporte de Ânions/genética , Genoma de Planta/genética , Canais Iônicos/genética , Proteínas de Plantas/genética , Produtos do Tabaco , Tabaco/genética , Transcriptoma/genética , Proteínas de Transporte de Ânions/metabolismo , Canais Iônicos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , RNA Longo não Codificante/genética , RNA de Plantas/genética , Tabaco/metabolismo , Fatores de Transcrição/genética
18.
New Phytol ; 232(1): 332-344, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34171146

RESUMO

Plants have developed tissue-specific defense strategies in response to various herbivores with different feeding habits. Although defense responses to leaf-chewing insects have been well studied, little is known about stem-specific responses, particularly in the pith, to stem-boring herbivores. To understand the stem-specific defense, we first conducted a comparative transcriptomic analysis of the wild tobacco Nicotiana attenuata before and after attack by the leaf-chewing herbivore Manduca sexta and the stem borer Trichobaris mucorea. When the stem-boring herbivore attacked, lignin-associated genes were upregulated specifically in the inner parenchymal cells of the stem, the pith; lignin also accumulated highly in the attacked pith. Silencing the lignin biosynthetic gene cinnamyl alcohol dehydrogenase enhanced the performance of the stem-boring herbivore but had no effect on the growth of the leaf-chewing herbivore. Two-dimensional nuclear magnetic resonance results revealed that lignified pith contains feruloyltyramine as an unusual lignin component in the cell wall, as a response against stem-boring herbivore attack. Pith-specific lignification induced by the stem-boring herbivore was modulated by both jasmonate and ethylene signaling. These results suggest that lignin provides a stem-specific inducible barrier, protecting plants against stem-boring insects.


Assuntos
Manduca , Gorgulhos , Animais , Regulação da Expressão Gênica de Plantas , Herbivoria , Tabaco/genética
19.
New Phytol ; 232(1): 356-371, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34185326

RESUMO

Virus-induced gene silencing (VIGS) can be harnessed to sequence-specifically degrade host transcripts and induce heritable epigenetic modifications referred to as virus-induced post-transcriptional gene silencing (ViPTGS) and virus-induced transcriptional gene silencing (ViTGS), respectively. Both ViPTGS and ViTGS enable manipulation of endogenous gene expression without the need for transgenesis. Although VIGS has been widely used in many plant species, it is not always uniform or highly efficient. The efficiency of VIGS is affected by developmental, physiological and environmental factors. Here, we use recombinant Tobacco rattle viruses (TRV) to study the effect of temperature on ViPTGS and ViTGS using GFP as a reporter gene of silencing in N. benthamiana 16c plants. We found that unlike ViPTGS, ViTGS was impaired at high temperature. Using a novel mismatch-small interfering RNA (siRNA) tool, which precisely distinguishes virus-derived (primary) from target-generated (secondary) siRNAs, we demonstrated that the lack of secondary siRNA production/amplification was responsible for inefficient ViTGS at 29°C. Moreover, inefficient ViTGS at 29°C inhibited the transmission of epigenetic gene silencing to the subsequent generations. Our finding contributes to understanding the impact of environmental conditions on primary and secondary siRNA production and may pave the way to design/optimize ViTGS for transgene-free crop improvement.


Assuntos
Vírus de Plantas , Vírus de DNA , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vírus de Plantas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Temperatura , Tabaco/genética
20.
J Plant Physiol ; 263: 153452, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34098414

RESUMO

In plants, jasmonate ZIM-domain proteins (JAZs) act as critical regulators, interacting physically with transcription factors (TFs) and other transcriptional regulators to modulate jasmonate (JA)-responsive gene expression and participate in crosstalk with other hormone signalling pathways. Identifying novel JAZ-interacting proteins will provide new insights into JA signalling cascades in plants. Here, we performed yeast two-hybrid screening to identify 70 NtJAZ1-interacting proteins, including an A/T-rich interaction domain containing protein 1 (NtAIDP1) from JA-treated tobacco Bright Yellow-2 (BY-2) cells. NtAIDP1 is localised in the nucleus and interacts with NtJAZ1 via its C-terminal heat shock protein 20 (HSP) domain. Aside from NtJAZ1, NtAIDP1 also interacts with other JA-inducible NtJAZs, including NtJAZ2b, NtJAZ2b.2, NtJAZ5, NtJAZ7, NtJAZ11 and NtJAZ12, but not with NtJAZ3, NtJAZ3b or NtJAZ10, and interacts with NtNINJA, NtDELLA1 and NtDELLA2 in the yeast two-hybrid assay. Furthermore, NtAIDP1 binds to the AT-rich region of the GAG fragment of the putrescine N-methyltransferase 1a (NtPMT1a) promoter and activates the transcriptional activity of the GAG fragment, whereas NtMYC2a interacts with and competitively inhibits the transactivational activity of NtAIDP1 in Arabidopsis mesophyll protoplasts. Overexpression of NtAIDP1 promotes the transcription of NtPDF1.2 and NtJAZ1, but has little effect on the expression of NtPMT1a, quinolinate phosphoribosyltransferase 2 (NtQPT2), and NtMYC2a in tobacco. These results indicate that NtAIDP1 is a new component of the JA signalling pathway and is involved in JA-regulated gene expression.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Tabaco/genética , Tabaco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas
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