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1.
Plant Sci ; 286: 1-6, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300135

RESUMO

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.


Assuntos
Adaptação Fisiológica/efeitos da radiação , Arabidopsis/metabolismo , Oryza/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Oryza/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Protoplastos/metabolismo , Protoplastos/efeitos da radiação
2.
Plant Sci ; 285: 44-54, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203893

RESUMO

Although the involvement of ROS (reactive oxygen species) in leaf senescence is well known, the factors governing this accumulation of ROS are not fully characterized. In this study, analysis of transgenic overexpressing and knock out lines of AtWDS1 (encoding a WD repeat protein), indicates that AtWDS1 negatively regulates age-dependent and dark-induced leaf senescence. Furthermore, we observed ROS accumulation and altered tolerance of oxidative stress in atwds1 plants, as well as upregulated expression of oxidative stress-responsive genes. The location of an EGFP-AtWDS1 fusion protein in the nucleus of transformed cells and plants indicates that AtWDS1 is a nuclear protein, and, using a Dual-Luciferase assay, we showed that AtWDS1 can act as a transcription activator. However, the lack of a nuclear localization sequence in AtWDS1 suggests that its presence in the nucleus must depend on interactions with other proteins. Indeed, we found that AtWDS1 interacts directly with AtRanBPM, and that mutation of the AtRanBPM gene results in partial mislocalization of AtWDS1 in the cytoplasm. Together, these results suggest a role for AtWDS1 as a novel modulator of redox homeostasis, which responds to developmental and stress signals to regulate leaf senescence.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/fisiologia , Clorofila/metabolismo , Escuridão , Microscopia Confocal , Folhas de Planta/fisiologia , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma
3.
Gene ; 709: 8-16, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132514

RESUMO

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.


Assuntos
Ascomicetos/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Fúngico , Protoplastos/metabolismo , Transformação Genética , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/genética
4.
Plant Physiol Biochem ; 141: 60-72, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128564

RESUMO

The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH) and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC) assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L) to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.


Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Iridaceae/metabolismo , Proantocianidinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Iridaceae/genética , Mutação , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Protoplastos/metabolismo , Tricomas/metabolismo , Repetições WD40
5.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987084

RESUMO

Increasing usage of gold nanoparticles (AuNPs) in different industrial areas inevitably leads to their release into the environment. Thus, living organisms, including plants, may be exposed to a direct contact with nanoparticles (NPs). Despite the growing amount of research on this topic, our knowledge about NPs uptake by plants and their influence on different developmental processes is still insufficient. The first physical barrier for NPs penetration to the plant body is a cell wall which protects cytoplasm from external factors and environmental stresses. The absence of a cell wall may facilitate the internalization of various particles including NPs. Our studies have shown that AuNPs, independently of their surface charge, did not cross the cell wall of Arabidopsis thaliana (L.) roots. However, the research carried out with using light and transmission electron microscope revealed that AuNPs with different surface charge caused diverse changes in the root's histology and ultrastructure. Therefore, we verified whether this is only the wall which protects cells against particles penetration and for this purpose we used protoplasts culture. It has been shown that plasma membrane (PM) is not a barrier for positively charged (+) AuNPs and negatively charged (-) AuNPs, which passage to the cell.


Assuntos
Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Protoplastos/metabolismo , Arabidopsis/ultraestrutura , Parede Celular/metabolismo , Nanopartículas Metálicas/ultraestrutura , Raízes de Plantas/ultraestrutura , Protoplastos/citologia , Protoplastos/ultraestrutura , Propriedades de Superfície
6.
J Biosci Bioeng ; 128(2): 129-134, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30824179

RESUMO

Aspergillus luchuensis NBRC4314 recently underwent genome sequencing. We have not used the frequently used protoplast-polyethylene glycol (PEG) method but have used agrobacterium-mediated transformation (AMT) to genetically engineer this strain because it was difficult to generate protoplasts using commercial cell wall lytic enzymes. In this study, we initially investigated the various conditions for protoplast formation in A. luchuensis. We found that A. luchuensis protoplasts could be generated using a minimal medium for the preculture medium, a static culture for the preculture condition, and Yatalase and α-1,3-glucanase as cell-wall lytic enzymes. These protoplasts could then be transformed with the protoplast-PEG method. Because α-1,3-glucanase was needed to form protoplasts in A. luchuensis, we investigated the role of the α-1,3-glucan synthase gene agsE in protoplast formation, one of five α-1,3-glucan synthase genes in A. luchuensis and a homolog of the major α-1,3-glucan synthase agsB in Aspergillus nidulans. We disrupted agsE in A. luchuensis (ΔagsE) with AMT and found that protoplast formation in ΔagsE was comparable with protoplast formation in Aspergillus oryzae with Yatalase. The ΔagsE protoplasts were also competent for transformation with the protoplast-PEG method. Hence, agsE appears to inhibit protoplast formation in A. luchuensis.


Assuntos
Aspergillus oryzae/citologia , Aspergillus oryzae/genética , Glucosiltransferases/genética , Protoplastos/metabolismo , Transformação Genética , Aspergillus nidulans/genética
7.
Plant Physiol ; 179(4): 1444-1456, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718350

RESUMO

Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants. Here, we describe the use of a commercially available droplet-based microfluidics platform for high-throughput scRNA-seq to obtain single-cell transcriptomes from protoplasts of more than 10,000 Arabidopsis (Arabidopsis thaliana) root cells. We find that all major tissues and developmental stages are represented in this single-cell transcriptome population. Further, distinct subpopulations and rare cell types, including putative quiescent center cells, were identified. A focused analysis of root epidermal cell transcriptomes defined developmental trajectories for individual cells progressing from meristematic through mature stages of root-hair and nonhair cell differentiation. In addition, single-cell transcriptomes were obtained from root epidermis mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes. Overall, this study demonstrates the feasibility and utility of scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.


Assuntos
Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Análise de Célula Única , Transcriptoma , Arabidopsis/citologia , Estudos de Viabilidade , Epiderme Vegetal/metabolismo , Raízes de Plantas/citologia , Protoplastos/metabolismo , Análise de Sequência de RNA
8.
Nat Nanotechnol ; 14(5): 456-464, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30804481

RESUMO

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.


Assuntos
Técnicas de Transferência de Genes , Gossypium/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Transgenes , Triticum/genética , Gossypium/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Triticum/metabolismo
9.
Plant Mol Biol ; 100(1-2): 47-58, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30783952

RESUMO

KEY MESSAGE: The two predicted WD40 propellers on TOPLESS function as protein-protein interaction domains. The 1st WD40 propeller mediates interaction with RAV1, and the 2nd WD40 propeller mediates interaction with VRN5. The TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressor family proteins are known to interact with a wide variety of proteins including transcription factors, Mediator subunits, histone deacetylases, and histone tails. Through these interactions, TPL/TPR act to repress transcription in an increasingly diverse array of plant pathways. Proteins that bind TPL/TPR typically contain one or more Repression Domains (RDs) that mediate the interaction. For example, the well-characterized Ethylene response factor-associated Amphiphilic Repression (EAR) motif is known to facilitate interaction by binding the TOPLESS Domain (TPD) located in the N-terminus. Here we show that in yeast two-hybrid assays, the non-EAR protein, Related to ABI3/VP1-1 (RAV1), binds a novel region located within the first nine WD40-repeats of TPL. Protein modeling and in silico analysis suggest that these nine WD40 repeats may form the first of two WD40 propellers located on C-terminus of TPL. The interaction between RAV1 and the 1st WD40 propeller is conserved with another RAV family member, TEMPRANILLO1 (TEM1) and is mediated by the B3 Repression Domain (BRD) located on both RAV1 and TEM1. Also, the predicted 2nd WD40 propeller was shown in yeast cells to bind Vernalization 5 (VRN5), which contains several unconfirmed partial RDs. Furthermore, we demonstrate that the 1st WD40 propeller of TPL can form a complex with RAV1 both in yeast and in Arabidopsis protoplasts.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Correpressoras/química , Proteínas Correpressoras/metabolismo , Repetições WD40 , Arabidopsis/metabolismo , Modelos Biológicos , Ligação Proteica , Protoplastos/metabolismo , Relação Estrutura-Atividade
10.
Int J Mol Sci ; 20(2)2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30634635

RESUMO

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC⁻MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV⁻host interaction that might contribute to the symptom development.


Assuntos
Cloroplastos/metabolismo , Oryza/metabolismo , Oryza/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Proteoma , Proteômica , Protoplastos/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Ontologia Genética , Fenótipo , Proteômica/métodos , Espectrometria de Massas em Tandem
11.
Methods Mol Biol ; 1917: 355-363, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610649

RESUMO

Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here we describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments.


Assuntos
Protoplastos/metabolismo , Tabaco/genética , Robótica , Transformação Genética/genética
12.
Ecotoxicol Environ Saf ; 170: 488-494, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553927

RESUMO

The aim of this article was to evaluate the viability of maize protoplasts, cell wall regeneration, Cd uptake by protoplasts, and the impact of silicon under cadmium cations (Cd) stress in two maize hybrids with contrasting tolerances to Cd toxicity. The differences in protoplast viability between the sensitive (Novania) and tolerant (Almansa) hybrids were noticeable even at the beginning of culture. The percentage of living protoplasts in the presence of Cd was higher in the tolerant hybrid. In both hybrids, Si supplementation significantly increased the viability of protoplasts exposed to Cd. The percentage of protoplasts with regenerated cell walls gradually increased in both hybrids and by the end of the culture it had reached almost identical values. Differences were observed during the first four days, when a lag phase occurred in the protoplasts of the sensitive hybrid accompanied by a rapid decrease in protoplast viability in all the variants tested. The addition of Si increased the cell wall regeneration compared with the Cd variant in both hybrids. The Cd content was higher in the tolerant hybrid than in the sensitive one during the first four days and declined on the seventh day. This may be connected with the increasing intensity of cell wall formation from the fourth up to the seventh day. The addition of Si decreased the Cd uptake into protoplasts of both hybrids. Despite the higher content of Cd, the protoplasts of the tolerant hybrid showed higher viability, obviously indicating unequal mechanisms of Cd processing in studied hybrids. CAPSULE: Protoplasts of two maize hybrids were tested for their viability, regeneration, Cd-uptake and the mitigation of cadmium stress by silicon.


Assuntos
Cádmio/toxicidade , Protoplastos/efeitos dos fármacos , Silício/farmacologia , Poluentes do Solo/toxicidade , Zea mays/efeitos dos fármacos , Transporte Biológico , Cádmio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quimera , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Regeneração , Poluentes do Solo/metabolismo , Zea mays/citologia , Zea mays/metabolismo
13.
Colloids Surf B Biointerfaces ; 173: 899-905, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30551307

RESUMO

Foliar fertilization is becoming very popular since it is highly efficient, minimizes contamination, and is target oriented. However, the low mobility of several nutrients and low penetration through the leaf cuticle should be addressed, to establish an efficient leaf applied fertilizer. During the last few years, nanotechnologies - such as nanocarriers - have been studied with regard to improving the penetration and delivery of compounds. This work describes the use of membrane vesicles obtained from Brassica oleracea L. as nanobiocarriers of Zn and the evaluation of their potential as a foliar fertilizer, also in Brassica. The results show a high Zn encapsulation efficiency and high delivery into protoplasts. Also, the foliar fertilization experiments demonstrated a very effective system of Zn nanofertilization.


Assuntos
Materiais Biocompatíveis/química , Fertilizantes , Nanocápsulas/química , Nanopartículas/química , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Zinco/química , Brassica , Membrana Celular/metabolismo , Quelantes/química , Luz , Lipídeos/química , Oryza , Tamanho da Partícula , Fotossíntese , Ligação Proteica , Espalhamento de Radiação , Sementes/metabolismo , Solo , Poluentes do Solo
14.
Methods Mol Biol ; 1864: 131-152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30415334

RESUMO

Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures of canola (Brassica napus). Key steps including enzymatic digestion for protoplast release, density gradient-based protoplast purification, PEG-mediated transfection, bead-type culturing (sea-plaque agarose and sodium alginate), and the recovery of putative transgenic canola plants are described. This method has been used for double-stranded DNA break-mediated genome editing and for the routine generation of stable transgenic canola events at commercial scale.


Assuntos
Brassica napus/genética , Plantas Geneticamente Modificadas/genética , Protoplastos/metabolismo , Transfecção/métodos , Brassica napus/metabolismo , Quebras de DNA de Cadeia Dupla , Edição de Genes/instrumentação , Edição de Genes/métodos , Plantas Geneticamente Modificadas/metabolismo , Polietilenoglicóis/química , Sementes , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos , Transfecção/instrumentação
15.
J Vis Exp ; (142)2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30582588

RESUMO

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Cloroplastos/fisiologia , Protoplastos/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico
16.
Plant Physiol Biochem ; 130: 445-454, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30077920

RESUMO

Soil acidity, associated with aluminium (Al) toxicity and low phosphorus (P) availability, is considered the most important problem for agricultural production. Even though the Al-P interaction has been widely investigated, the impact of P-nutrition on Al-toxicity still remains controversial and poorly understood. To elucidate further insights into the underlying mechanisms of this interaction in ryegrass (Lolium perenne L.), P uptake, antioxidant responses and the gene expression of phosphate transporters were determined. Two ryegrass cultivars with different Al resistances, the Al-tolerant Nui cultivar and the Al-sensitive Expo cultivar were hydroponically grown under low (16 µM) and optimal (100 µM) P doses for 16 days. After P treatments, plants were exposed to Al doses (0 and 200 µM) under acidic conditions (pH 4.8) for 24 h. Al and P accumulation were higher in the roots of Nui than that of Expo. Moreover, lower Al accumulation was found in shoots of Nui independent of P supplies. Oxidative stress induced by Al-toxicity and P-deficiency was more severe in the Al-sensitive Expo. Expression levels of L. perenne phosphate transporters were higher in Nui than they were in Expo. While LpPHT1 expression was up-regulated by P deficiency and Al toxicity in both cultivars, LpPHT4 expression only increased in the Al-tolerant cultivar. This report shows that the higher Al-tolerance of Nui can be attributed to a greater antioxidant system under both P conditions. The observation of higher P and Al accumulation in roots of Nui might indicate that the Al-tolerance of Nui is a consequence of Al immobilization by P mediated by the high expression of phosphate transporters.


Assuntos
Alumínio/toxicidade , Antioxidantes/metabolismo , Lolium/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/deficiência , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Estresse Oxidativo , Peroxidase/metabolismo , Proteínas de Transporte de Fosfato/fisiologia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Protoplastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , Superóxido Dismutase/metabolismo , Transcriptoma , Regulação para Cima
17.
J Vis Exp ; (138)2018 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-30148492

RESUMO

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.


Assuntos
Microscopia Confocal/métodos , Peptídeos/metabolismo , Protoplastos/metabolismo , Esferoplastos/metabolismo , Protoplastos/citologia , Esferoplastos/citologia
18.
Molecules ; 23(6)2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29794975

RESUMO

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/µg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Protoplastos/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Transformação Genética , Técnicas de Transferência de Genes , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polietilenoglicóis , Protoplastos/enzimologia , Saccharomycetales/genética , Saccharomycetales/metabolismo , Temperatura Ambiente , Triticum/microbiologia
19.
Planta ; 248(3): 571-578, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29799081

RESUMO

MAIN CONCLUSION: Our transient gene expression analyses in Arabidopsis protoplasts support the view that CK2αs and CK2ßs positively and negatively modulate ABRE-dependent gene expression, respectively. The phytohormone abscisic acid (ABA) regulates the expression of thousands of genes via ABA-responsive elements (ABREs), and has a crucial role in abiotic stress response. Casein kinase II (CK2), a conserved Ser/Thr protein kinase in eukaryotes, is essential for plant viability. Although the CK2 has been known as a tetrameric holoenzyme comprised of two catalytic α and two regulatory ß subunits, each of the two types of subunits has been proposed to have independent functions. The Arabidopsis genome encodes four α subunits (CK2α1, CK2α2, CK2α3, CK2α4) and four ß subunits (CK2ß1, CK2ß2, CK2ß3, CK2ß4). There is a growing body of evidence linking CK2 to ABA signaling and abiotic stress responses. However, the roles of each CK2 subunit in ABA signaling remain largely elusive. Using the transient expression system with the core ABA signaling components in Arabidopsis leaf mesophyll protoplasts, we show here that CK2α1 and CK2α2 (CK2α1/2) positively modulate ABRE-dependent gene expression as ABA signal output in ABA signaling, whereas all four CK2ßs negatively modulate the ABRE-dependent gene expression mediated by subclass III SnRK2-AREB/ABF pathway and by CK2α1/2. These data indicate that CK2α1/2 and CK2ßs positively and negatively modulate ABA signal output, respectively, suggesting that the quantitative balance of CK2 subunits determines the ABA signal output in plants. Given that CK2s act as pleiotropic enzymes involved in multiple developmental and stress-responsive processes, our findings suggest that CK2 subunits may be involved in integration and coordination of ABA-dependent and -independent signaling.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Caseína Quinase II/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Caseína Quinase II/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Transdução de Sinais
20.
Planta ; 248(1): 231-241, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29700611

RESUMO

MAIN CONCLUSION: Rice MTP11 is the trans-Golgi-localized transporter that is involved in Mn tolerance with MTP8.1, and it is required for normal fertility. Rice (Oryza sativa L.) is one of the most manganese (Mn)-tolerant species, and it is able to accumulate high levels of this metal in the leaves without showing toxic symptoms. The metal tolerance protein 8.1 (MTP8.1), a member of the Mn-cation diffusion facilitator (CDF) family, has been shown to play a central role in high Mn tolerance by sequestering Mn into vacuoles. Recently, rice MTP11 was identified as an Mn transporter that is localized to Golgi-associated compartments, but its exact role in Mn tolerance in planta has not yet been understood. Here, we investigated the role of MTP11 in rice Mn tolerance using knockout lines. Old leaves presented higher levels of constitutively expressed MTP11 than other tissues and MTP11 expression was also found in reproductive organs. Fused MTP11:green fluorescent protein was co-localized to trans-Golgi markers and differentiated from other Golgi-associated markers. Knockout of MTP11 in wild-type rice did not affect tolerance and accumulation of Mn and other heavy metals, but knockout in the mtp8.1 mutant showed exacerbated Mn sensitivity at the vegetative growth stage. Knockout of MTP11 alone resulted in decreased grain yield and fertility at the reproductive stage. Thus, MTP11 is a trans-Golgi localized transporter for Mn, which plays a role in Mn tolerance through intracellular Mn compartmentalization. It is also required for maintaining high fertility in rice.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Manganês/toxicidade , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Transporte de Cátions/genética , Fertilidade , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Manganês/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
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