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1.
Proc Natl Acad Sci U S A ; 117(40): 25150-25158, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968023

RESUMO

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.


Assuntos
Endocitose/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Proteínas R-SNARE/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/genética , Clatrina/metabolismo , Eucariotos/genética , Exocitose/genética , Regulação da Expressão Gênica de Plantas/genética , Células Vegetais/metabolismo , Domínios Proteicos/genética
2.
Proc Natl Acad Sci U S A ; 117(33): 20316-20324, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737163

RESUMO

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucanos/biossíntese , Glucosiltransferases/metabolismo , Células Vegetais/metabolismo , Xilanos/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Mutação , Filogenia
3.
PLoS One ; 15(8): e0237478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853208

RESUMO

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-ß-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94-0.97) as compared to regression models (0.16-0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 µg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.


Assuntos
Técnicas de Cultura de Células/métodos , Corylus/química , Paclitaxel/biossíntese , Algoritmos , Corylus/metabolismo , Fungos/química , Fungos/metabolismo , Modelos Lineares , Células Vegetais/química , Células Vegetais/metabolismo , beta-Ciclodextrinas/química
4.
Ecotoxicol Environ Saf ; 202: 110904, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800239

RESUMO

Cation diffusion facilitators (CDFs) play central roles in metal homeostasis and tolerance in plants, but the specific functions of Camellia sinensis CDF-encoding genes and the underlying mechanisms remain unknown. Previously, transcriptome sequencing results in our lab indicated that the expression of CsMTP8.2 in tea plant shoots was down-regulated exposed to excessive amount of Mn2+ conditions. To elucidate the possible mechanisms involved, we systematically identified 13 C. sinensis CsMTP genes from three subfamilies and characterized their phylogeny, structures, and the features of the encoded proteins. The transcription of CsMTP genes was differentially regulated in C. sinensis shoots and roots in responses to high concentrations of Mn, Zn, Fe, and Al. Differences in the cis-acting regulatory elements in the CsMTP8.1 and CsMTP8.2 promoters suggested the expression of these two genes may be differentially regulated. Transient expression analysis indicated that CsMTP8.2 was localized to the plasma membrane in tobacco and onion epidermal cells. Moreover, when heterologously expressed in yeast, CsMTP8.2 conferred tolerance to Ni and Mn but not to Zn. Additionally, heterologous expression of CsMTP8.2 in Arabidopsis thaliana revealed that CsMTP8.2 positively regulated the response to manganese toxicity by decreasing the accumulation of Mn in plants. However, there was no difference in the accumulation of other metals, including Cu, Fe, and Zn. These results suggest that CsMTP8.2 is a Mn-specific transporter that contributes to the efflux of excess Mn2+ from plant cells.


Assuntos
Camellia sinensis/genética , Manganês/toxicidade , Poluentes do Solo/toxicidade , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Manganês/metabolismo , Filogenia , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Saccharomyces cerevisiae/metabolismo , Chá
5.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA Replicase/genética , RNA de Cadeia Dupla/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
6.
PLoS Comput Biol ; 16(7): e1007523, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687508

RESUMO

Coordination of fate transition and cell division is crucial to maintain the plant architecture and to achieve efficient production of plant organs. In this paper, we analysed the stem cell dynamics at the shoot apical meristem (SAM) that is one of the plant stem cells locations. We designed a mathematical model to elucidate the impact of hormonal signaling on the fate transition rates between different zones corresponding to slowly dividing stem cells and fast dividing transit amplifying cells. The model is based on a simplified two-dimensional disc geometry of the SAM and accounts for a continuous displacement towards the periphery of cells produced in the central zone. Coupling growth and hormonal signaling results in a nonlinear system of reaction-diffusion equations on a growing domain with the growth rate depending on the model components. The model is tested by simulating perturbations in the level of key transcription factors that maintain SAM homeostasis. The model provides new insights on how the transcription factor HECATE is integrated in the regulatory network that governs stem cell differentiation.


Assuntos
Diferenciação Celular/fisiologia , Modelos Biológicos , Células Vegetais , Reguladores de Crescimento de Planta/fisiologia , Transdução de Sinais/fisiologia , Biologia Computacional , Simulação por Computador , Meristema/citologia , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Meristema/fisiologia , Células Vegetais/metabolismo , Células Vegetais/fisiologia
7.
Food Chem ; 330: 127318, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569935

RESUMO

The objective of this study was to determine the biophysical properties of buckwheat (BW) endosperm and their influences on detachment of intact cells, starch gelatinization and digestibility. The intact cells were isolated from BW kernels by dry milling and sieving. The microscopy and texture analysis showed intact endosperm cells could be detached easily due to the fragile structure and low hardness of BW endosperm. More than 70% intact cells were found in commercial light flour. The starch granules entrapped in intact cells exhibited a delay gelatinization and restricted swelling behavior (2-3 â„ƒ higher onset gelatinization temperature than isolated starch). Starch in BW flour had a markedly lower extent of digestion compared to the broken cells and isolated starch. This study provided a new mechanistic understanding of low glycemic index of BW food, and could guide the processing of BW flour to retain slow digestion properties.


Assuntos
Endosperma/citologia , Fagopyrum/citologia , Fagopyrum/metabolismo , Farinha , Amido/farmacocinética , Culinária , Digestão , Endosperma/química , Endosperma/metabolismo , Fagopyrum/química , Farinha/análise , Gelatina , Índice Glicêmico , Tamanho da Partícula , Células Vegetais/química , Células Vegetais/metabolismo , Amido/química , Temperatura
8.
PLoS One ; 15(6): e0234011, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484825

RESUMO

The tomato AGC protein kinase Adi3 is a Ser/Thr kinase that functions as a negative regulator of programmed cell death through cell death suppression (CDS) activity in the nucleus. In this study, to understand the mechanism of Adi3 CDS, peptide microarrays containing random Ser- and Thr-peptide phosphorylation substrates were used to screen for downstream phosphorylation substrates. In the microarray phosphorylation assay, Adi3 showed promiscuous kinase activity more toward Ser-peptides compared to Thr-peptides, and a preference for aromatic and cyclic amino acids on both Ser- and Thr-peptides was seen. The 63 highest phosphorylated peptide sequences from the Ser-peptide microarray were selected as queries for a BLAST search against the tomato proteome. As a result, 294 candidate nuclear Adi3 substrates were selected and categorized based on their functions. Many of these proteins were classified as DNA/RNA polymerases or regulators involved in transcription and translation events. The list of potential Adi3 substrates was narrowed to eleven and four candidates were tested for phosphorylation by Adi3. Two of these candidates, RNA polymerase II 2nd largest subunit (RPB2) and the pathogen defense related transcription factor Pti5, were confirmed as Adi3 phosphorylation substrates by in vitro kinase assays. Using a mutational approach two residues, Thr675 and Thr676, were identified as Adi3 phosphorylation sites on RPB2. This study provides the foundation for understanding Adi3 CDS mechanisms in the nucleus as well as other cellular functions.


Assuntos
Morte Celular/genética , Peptídeos/genética , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Sequência de Aminoácidos , Núcleo Celular/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Análise em Microsséries , Mutação/genética , Fosforilação/genética , Proteínas Quinases/genética
9.
Nat Chem Biol ; 16(7): 740-748, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424305

RESUMO

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.


Assuntos
Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glicosiltransferases/genética , Proteínas de Plantas/genética , Saponinas/biossíntese , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Retículo Endoplasmático/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Spinacia oleracea/genética
10.
PLoS One ; 15(5): e0232029, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374732

RESUMO

BACKGROUND: Translationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce. METHODOLOGY: To carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins. RESULTS: A total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner. CONCLUSIONS: Our analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Evolução Molecular , Especiação Genética , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/química , Sequência Conservada , Células Eucarióticas/classificação , Células Eucarióticas/metabolismo , Fungos/classificação , Fungos/genética , Humanos , Invertebrados/classificação , Invertebrados/genética , Mamíferos/classificação , Mamíferos/genética , Simulação de Acoplamento Molecular , Filogenia , Células Vegetais/classificação , Células Vegetais/metabolismo , Células Procarióticas/classificação , Células Procarióticas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade da Espécie
11.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269127

RESUMO

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.


Assuntos
Interações Hospedeiro-Patógeno/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tombusvirus/genética , Replicação Viral/genética , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Protoplastos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Replicase/genética , RNA Replicase/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-32335386

RESUMO

Boron (B) deficiency is frequently observed in citrus orchards as a major cause for loss of productivity and quality. The structural and morphological responses of roots to B deficiency have been reported in some plants. The study was conducted to get novel information about the B-deficient-induced cellular injuries and target secondary metabolites in the shikimate pathway. Fluorescent vital staining, paraffin section, transmission electron microscopy (TEM) and target metabolomics were to investigate the responses of the cell viability and structure, and target aromatic metabolites in the shikimate pathway in B-deficient trifoliate orange roots. Boron deprivation-induced ROS accumulation accelerated the membrane peroxidation, resulting in weakened cell vitality and cell rupture in roots. In addition, B deficiency increased phenylalanine (Phe), tyrosine (Try) in roots, thereby promoting the biosynthesis of salicylic acid, caffeic acid and ferulic acid. B-starvation up-regulated salicylic acid and lignin while reduced 3-indoleacetic acid (IAA) content. These adverse effects might be involved in the structural and morphological changes in B-deficient roots. What is more, the results provide a new insight into the mechanism of B deficiency-induced structural damage and elongation inhibition on roots.


Assuntos
Boro , Células Vegetais , Poncirus , Boro/deficiência , Células Vegetais/metabolismo , Células Vegetais/patologia , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Poncirus/química , Poncirus/metabolismo , Ácido Chiquímico/metabolismo
13.
Nat Commun ; 11(1): 1660, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245953

RESUMO

Warm temperature is postulated to induce plant thermomorphogenesis through a signaling mechanism similar to shade, as both destabilize the active form of the photoreceptor and thermosensor phytochrome B (phyB). At the cellular level, shade antagonizes phyB signaling by triggering phyB disassembly from photobodies. Here we report temperature-dependent photobody localization of fluorescent protein-tagged phyB (phyB-FP) in the epidermal cells of Arabidopsis hypocotyl and cotyledon. Our results demonstrate that warm temperature elicits different photobody dynamics than those by shade. Increases in temperature from 12 °C to 27 °C incrementally reduce photobody number by stimulating phyB-FP disassembly from selective thermo-unstable photobodies. The thermostability of photobodies relies on phyB's photosensory module. Surprisingly, elevated temperatures inflict opposite effects on phyB's functions in the hypocotyl and cotyledon despite inducing similar photobody dynamics, indicative of tissue/organ-specific temperature signaling circuitry either downstream of photobody dynamics or independent of phyB. Our results thus provide direct cell biology evidence supporting an early temperature signaling mechanism via dynamic assembly/disassembly of individual photobodies possessing distinct thermostabilities.


Assuntos
Proteínas de Arabidopsis/metabolismo , Estruturas do Núcleo Celular/metabolismo , Células Fotorreceptoras/metabolismo , Fitocromo B/metabolismo , Temperatura , Arabidopsis/metabolismo , Cotilédone/citologia , Cotilédone/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/citologia , Hipocótilo/metabolismo , Luz , Células Vegetais/metabolismo , Epiderme Vegetal/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
14.
Planta ; 251(4): 82, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32189080

RESUMO

MAIN CONCLUSION: The review provides information on the mechanisms underlying the development of spontaneous and pathogen-induced tumors in higher plants. The activation of meristem-specific regulators in plant tumors of various origins suggests the meristem-like nature of abnormal plant hyperplasia. Plant tumor formation has more than a century of research history. The study of this phenomenon has led to a number of important discoveries, including the development of the Agrobacterium-mediated transformation technique and the discovery of horizontal gene transfer from bacteria to plants. There are two main groups of plant tumors: pathogen-induced tumors (e.g., tumors induced by bacteria, viruses, fungi, insects, etc.), and spontaneous ones, which are formed in the absence of any pathogen in plants with certain genotypes (e.g., interspecific hybrids, inbred lines, and mutants). The causes of the transition of plant cells to tumor growth are different from those in animals, and they include the disturbance of phytohormonal balance and the acquisition of meristematic characteristics by differentiated cells. The aim of this review is to discuss the mechanisms underlying the development of most known examples of plant tumors.


Assuntos
Tumores de Planta/microbiologia , Animais , Bactérias/metabolismo , Fungos/metabolismo , Interações Hospedeiro-Patógeno , Insetos/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/microbiologia , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Reguladores de Crescimento de Planta/metabolismo , Vírus/metabolismo
15.
Crit Rev Biotechnol ; 40(4): 443-458, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32178548

RESUMO

For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the lab- or industrial-scale production of plant secondary metabolites are specified.


Assuntos
Reatores Biológicos , Engenharia Metabólica/métodos , Células Vegetais/metabolismo , Metabolismo Secundário , Sistemas CRISPR-Cas , Edição de Genes , Plantas/genética , Plantas/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-32120144

RESUMO

Elicited broccoli suspension-cultured cells (SCC) provide a useful system for obtaining bioactive compounds, including glucosinolates (GS) and phenolic compounds (PCs). In this work, coronatine (Cor) and methyl jasmonate (MJ) were used to increase the bioactive compound production in broccoli SCC. Although the use of Cor and MJ in secondary metabolite production has already been described, information concerning how elicitors affect cell metabolism is scarce. It has been suggested that Cor and MJ trigger defence reactions affecting the antioxidative metabolism. In the current study, the concentration of 0.5 µM Cor was the most effective treatment for increasing both the total antioxidant capacity (measured as ferulic acid equivalents) and glucosinolate content in broccoli SCC. The elicited broccoli SCC also showed higher polyphenol oxidase activity than the control cells. Elicitation altered the antioxidative metabolism of broccoli SCC, which displayed biochemical changes in antioxidant enzymes, a decrease in the glutathione redox state and an increase in lipid peroxidation levels. Furthermore, we studied the effect of elicitation on the protein profile and observed an induction of defence-related proteins. All of these findings suggest that elicitation not only increases bioactive compound production, but it also leads to mild oxidative stress in broccoli SCC that could be an important factor triggering the production of these compounds.


Assuntos
Acetatos/administração & dosagem , Aminoácidos/administração & dosagem , Antioxidantes/metabolismo , Brassica/metabolismo , Ciclopentanos/administração & dosagem , Glucosinolatos/metabolismo , Indenos/administração & dosagem , Oxilipinas/administração & dosagem , Compostos Fitoquímicos/metabolismo , Brassica/citologia , Ácidos Cumáricos/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glutationa/metabolismo , Peroxidação de Lipídeos , Células Vegetais/metabolismo , Reguladores de Crescimento de Planta
17.
Sci Rep ; 10(1): 3525, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103059

RESUMO

Asymmetric divisions maintain long-term stem cell populations while producing new cells that proliferate and then differentiate. Recent reports in animal systems show that divisions of stem cells can be uncoupled from their progeny differentiation, and the outcome of a division could be influenced by microenvironmental signals. But the underlying system-level mechanisms, and whether this dynamics also occur in plant stem cell niches (SCN), remain elusive. This article presents a cell fate regulatory network model that contributes to understanding such mechanism and identify critical cues for cell fate transitions in the root SCN. Novel computational and experimental results show that the transcriptional regulator SHR is critical for the most frequent asymmetric division previously described for quiescent centre stem cells. A multi-scale model of the root tip that simulated each cell's intracellular regulatory network, and the dynamics of SHR intercellular transport as a cell-cell coupling mechanism, was developed. It revealed that quiescent centre cell divisions produce two identical cells, that may acquire different fates depending on the feedback between SHR's availability and the state of the regulatory network. Novel experimental data presented here validates our model, which in turn, constitutes the first proposed systemic mechanism for uncoupled SCN cell division and differentiation.


Assuntos
Arabidopsis/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Modelos Biológicos , Células Vegetais/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/citologia , Raízes de Plantas/citologia
18.
Nature ; 578(7796): 577-581, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32076270

RESUMO

Hydrogen peroxide (H2O2) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues1-4. H2O2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H2O2 also exist on the cell surface remains unknown. In plant cells, H2O2 triggers an influx of Ca2+ ions, which is thought to be involved in H2O2 sensing and signalling. Here, by using forward genetic screens based on Ca2+ imaging, we isolated hydrogen-peroxide-induced Ca2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H2O2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H2O2-induced activation of Ca2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H2O2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cisteína/química , Cisteína/metabolismo , Ativação Enzimática , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Oxirredução , Células Vegetais/metabolismo , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética
19.
Proc Natl Acad Sci U S A ; 117(9): 5049-5058, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32051250

RESUMO

The coordinated redistribution of sugars from mature "source" leaves to developing "sink" leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) metabolic signaling network restricts PD transport in leaves. Genetic approaches and chemical or physiological treatments to either promote or disrupt TOR activity demonstrate that glucose-activated TOR decreases PD transport in leaves. We further found that TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and that this increase in TOR activity correlates with decreased rates of PD transport. We conclude that leaf cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Vegetais/metabolismo , Folhas de Planta/metabolismo , Plasmodesmos/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Metabolismo dos Carboidratos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Inativação Gênica , Folhas de Planta/crescimento & desenvolvimento , Transporte Proteico , Transdução de Sinais , Tabaco/genética , Tabaco/metabolismo
20.
Nat Commun ; 11(1): 958, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075974

RESUMO

The structural integrity of living plant cells heavily relies on the plant cell wall containing a nanofibrous cellulose skeleton. Hence, if synthetic plant cells consist of such a cell wall, they would allow for manipulation into more complex synthetic plant structures. Herein, we have overcome the fundamental difficulties associated with assembling lipid vesicles with cellulosic nanofibers (CNFs). We prepare plantosomes with an outer shell of CNF and pectin, and beneath this, a thin layer of lipids (oleic acid and phospholipids) that surrounds a water core. By exploiting the phase behavior of the lipids, regulated by pH and Mg2+ ions, we form vesicle-crowded interiors that change the outer dimension of the plantosomes, mimicking the expansion in real plant cells during, e.g., growth. The internal pressure enables growth of lipid tubules through the plantosome cell wall, which paves the way to the development of hierarchical plant structures and advanced synthetic plant cell mimics.


Assuntos
Células Artificiais/metabolismo , Materiais Biomiméticos/metabolismo , Parede Celular/metabolismo , Células Vegetais/metabolismo , Células Artificiais/citologia , Materiais Biomiméticos/química , Cápsulas/química , Cápsulas/metabolismo , Parede Celular/química , Parede Celular/ultraestrutura , Celulose/química , Microfluídica , Nanofibras/química , Ácido Oleico/química , Pectinas/química
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