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1.
Nucleic Acids Res ; 51(6): 2641-2654, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-36864547

RESUMO

Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.


Assuntos
Cromátides , Hordeum , Metáfase , Cromátides/química , Cromatina/genética , Cromossomos , Microscopia , Troca de Cromátide Irmã , Cromossomos de Plantas , Hordeum/citologia
2.
PLoS Genet ; 17(5): e1009292, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33970916

RESUMO

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.


Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Alelos , Sistemas CRISPR-Cas/genética , Divisão Celular , Hordeum/citologia , Mutação , Oryza/genética , Fenótipo , Células Vegetais , Folhas de Planta/citologia , Fatores de Tempo
3.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672992

RESUMO

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.


Assuntos
Cromossomos de Plantas/genética , Hordeum/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Cromossomos de Plantas/química , Cromossomos de Plantas/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Corantes Fluorescentes/química , Hordeum/citologia , Indóis/química , Metáfase/genética , Reprodutibilidade dos Testes
4.
Plant J ; 104(3): 752-767, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32799357

RESUMO

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.


Assuntos
Parede Celular/metabolismo , Glucanos/metabolismo , Glicosiltransferases/metabolismo , Hordeum/metabolismo , Oligossacarídeos/metabolismo , Xilanos/metabolismo , Ânions/metabolismo , Domínio Catalítico , Fluoresceínas/química , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/genética , Hordeum/citologia , Hordeum/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Família Multigênica , Oligossacarídeos/química , Pectinas/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Especificidade por Substrato , Ácidos Sulfônicos/química
5.
Int J Mol Sci ; 21(12)2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32575771

RESUMO

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.


Assuntos
Sulfato de Cobre/efeitos adversos , Desmetilação do DNA , Hordeum/citologia , Mutação , Nitrato de Prata/efeitos adversos , Ilhas de CpG , Meios de Cultura/efeitos adversos , Meios de Cultura/química , DNA de Plantas/efeitos dos fármacos , DNA de Plantas/genética , Epigênese Genética , Flores/citologia , Flores/genética , Haploidia , Hordeum/genética , Fatores de Tempo , Técnicas de Cultura de Tecidos
6.
Environ Geochem Health ; 42(1): 45-58, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30874936

RESUMO

Effects of Cu toxicity from contaminated soil were analysed in spring barley (Hordeum sativum distichum), a widely cultivated species in South Russia. In this study, H. sativum was planted outdoors in one of the most fertile soils-Haplic Chernozem spiked with high concentration of Cu and examined between the boot and head emergence phase of growth. Copper toxicity was observed to cause slow ontogenetic development of plants, changing their morphometric parameters (shape, size, colour). To the best of our knowledge, the ultrastructural changes in roots, stems and leaves of H. sativum induced by excess Cu were fully characterized for the first time using transmission electron microscopy. The plant roots were the most effected, showing degradation of the epidermis, reduced number of parenchyma cells, as well as a significant decrease in the diameter of the stele and a disruption and modification to its cell structure. The comparative analysis of the ultrastructure of control plants and plants exposed to the toxic effects of Cu has made it possible to reveal significant disruption of the integrity of the cell wall and cytoplasmic membranes in the root with deposition of electron-dense material. The changes in the ultrastructure of the main cytoplasmic organelles-endoplasmic reticulum, mitochondria, chloroplasts and peroxisomes-in the stem and leaves were found. The cellular Cu deposition, anatomical and ultrastructural modifications could mainly account for the primary impact points of metal toxicity. Therefore, this work extends the available knowledge of the mechanisms of the Cu effect tolerance of barley.


Assuntos
Cobre/toxicidade , Hordeum/efeitos dos fármacos , Poluentes do Solo/toxicidade , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Hordeum/anatomia & histologia , Hordeum/citologia , Hordeum/ultraestrutura , Microscopia Eletrônica de Transmissão , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/ultraestrutura , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Caules de Planta/citologia , Caules de Planta/efeitos dos fármacos , Caules de Planta/ultraestrutura , Federação Russa
7.
Plant J ; 98(6): 961-974, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31021020

RESUMO

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.


Assuntos
Variação Genética , Proteínas de Plantas/genética , Poaceae/genética , Alelos , Sequência de Aminoácidos , Evolução Biológica , Geografia , Haplótipos , Hordeum/citologia , Hordeum/genética , Imageamento por Ressonância Magnética , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Poaceae/citologia , Reprodução , Sementes/citologia , Sementes/genética , Alinhamento de Sequência , Especificidade da Espécie , Triticum/citologia , Triticum/genética
8.
Sci Rep ; 9(1): 3745, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842645

RESUMO

Amyloplasts are plant-specific organelles responsible for starch biosynthesis and storage. Inside amyloplasts, starch forms insoluble particles, referred to as starch grains (SGs). SG morphology differs between species and SG morphology is particularly diverse in the endosperm of Poaceae plants, such as rice (Oryza sativa) and barley (Hordeum vulgare), which form compound SGs and simple SGs, respectively. SG morphology has been extensively imaged, but the comparative imaging of amyloplast morphology has been limited. In this study, SG-containing amyloplasts in the developing endosperm were visualized using stable transgenic barley and rice lines expressing amyloplast stroma-targeted green fluorescent protein fused to the transit peptide (TP) of granule-bound starch synthase I (TP-GFP). The TP-GFP barley and rice plants had elongated amyloplasts containing multiple SGs, with constrictions between the SGs. In barley, some amyloplasts were connected by narrow protrusions extending from their surfaces. Transgenic rice lines producing amyloplast membrane-localized SUBSTANDARD STARCH GRAIN6 (SSG6)-GFP were used to demonstrate that the developing amyloplasts contained multiple compound SGs. TP-GFP barley can be used to visualize the chloroplasts in leaves and other plastids in pollen and root in addition to the endosperm, therefore it provides as a useful tool to observe diverse plastids.


Assuntos
Hordeum/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Plastídeos/metabolismo , Transaminases/metabolismo , Endosperma/citologia , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Hordeum/citologia , Hordeum/metabolismo , Imagem Molecular , Oryza/citologia , Oryza/metabolismo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
9.
Methods Mol Biol ; 1900: 153-166, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30460564

RESUMO

Microscopic investigations of biological objects are an integral part in plant research and most fields of life sciences. They allow the description of morphological, histological, and structural aspects of individual cells or tissues. Based on various cell biological tools and methods it is possible to characterize different plant genotypes or study their adaptation to changing environmental conditions. In combination with antibodies raised against specific antigens and epitopes light and electron microscopy enable investigation of the function of single genes/proteins in plant growth and development or their role related to abiotic or biotic stresses.Here, we describe sample preparation of barley roots for cell biological investigations using light and electron microscopy, to characterize morphological, structural, and functional aspects on root sections and the root surface.


Assuntos
Hordeum/imunologia , Hordeum/ultraestrutura , Microscopia Eletrônica/métodos , Raízes de Plantas/imunologia , Raízes de Plantas/ultraestrutura , Dessecação , Hordeum/citologia , Micro-Ondas , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Polimerização , Coloração e Rotulagem , Fixação de Tecidos
10.
Methods Mol Biol ; 1900: 167-179, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30460565

RESUMO

Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (Hordeum Vulgare) have provided new insights into the control of crossing over in large genome species. A major achievement in these studies has been the use of cytological procedures to follow meiotic events. This protocol provides detailed practical steps required to perform immunostaining of barley meiocytes (pollen mother cells) for confocal or structured illumination microscopy.


Assuntos
Hordeum/citologia , Microscopia/métodos , Pólen/citologia , Pareamento Cromossômico , Meiose
11.
Sci Rep ; 8(1): 9655, 2018 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-29941955

RESUMO

Waterlogging is one of the major abiotic stresses that affects barley production and yield quality. Proteomics techniques have been widely utilized to explore the mechanisms involved in the responses to abiotic stress. In this study, two barley genotypes with contrasting responses to waterlogging stress were analyzed with proteomic technology. The waterlogging treatment caused a greater reduction in biomass and photosynthetic performance in the waterlogging-sensitive genotype TF57 than that in the waterlogging-tolerant genotype TF58. Under waterlogging stress, 30, 30, 20 and 20 differentially expressed proteins were identified through tandem mass spectrometry analysis in the leaves, adventitious roots, nodal roots and seminal roots, respectively. Among these proteins, photosynthesis-, metabolism- and energy-related proteins were differentially expressed in the leaves, with oxygen-evolving enhancer protein 1, ATP synthase subunit and heat shock protein 70 being up-regulated in TF58. Pyruvate decarboxylase (PDC), 1-amino cyclopropane 1-carboxylic acid oxidase (ACO), glutamine synthetase (GS), glutathione S-transferases (GST) and beta-1, 3-glucanase in adventitious, nodal and seminal roots were more abundant in TF58 than those in TF57 under waterlogging stress. Ten representative genes were selected for validation by qRT-PCR in different genotypes with known waterlogging tolerance, and the expression levels of three candidate genes (PDC, ACO and GST) increased in the roots of all genotypes in response to the waterlogging stress. These three genes might play a significant role in the adaptation process of barley under waterlogging stress. The current results partially determined the mechanisms of waterlogging tolerance and provided valuable information for the breeding of barley with enhanced tolerance to waterlogging.


Assuntos
Hordeum/efeitos dos fármacos , Hordeum/fisiologia , Proteômica , Estresse Fisiológico/efeitos dos fármacos , Água/farmacologia , Hipóxia Celular , Relação Dose-Resposta a Droga , Genótipo , Hordeum/citologia , Hordeum/genética
12.
Plant J ; 93(6): 1062-1075, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29377449

RESUMO

Mixed-linkage (1,3;1,4)-ß-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-ß-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.


Assuntos
Brachypodium/metabolismo , Parede Celular/metabolismo , Complexo de Golgi/metabolismo , beta-Glucanas/metabolismo , Sequência de Aminoácidos , Brachypodium/citologia , Brachypodium/genética , Parede Celular/ultraestrutura , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Complexo de Golgi/ultraestrutura , Hordeum/citologia , Hordeum/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Homologia de Sequência de Aminoácidos
13.
New Phytol ; 218(3): 1127-1142, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28836669

RESUMO

The angiosperm embryo and endosperm are limited in space because they grow inside maternal seed tissues. The elimination of cell layers of the maternal seed coat by programmed cell death (PCD) could provide space and nutrition to the filial organs. Using the barley (Hordeum vulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains. The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains. The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals.


Assuntos
Apoptose , Cisteína Endopeptidases/metabolismo , Grão Comestível/anatomia & histologia , Hordeum/anatomia & histologia , Hordeum/citologia , Proteínas de Plantas/metabolismo , Sementes/citologia , Sementes/metabolismo , Apoptose/genética , Caspases/metabolismo , Contagem de Células , Endosperma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Tamanho do Órgão , Especificidade de Órgãos , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ploidias , Proteólise , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transcrição Gênica , Transcriptoma/genética
14.
Methods Mol Biol ; 1696: 57-79, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29086396

RESUMO

In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.


Assuntos
Hordeum/citologia , Proteínas Nucleares/análise , Proteômica/métodos , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Cromatografia Gasosa-Espectrometria de Massas , Hordeum/metabolismo , Proteínas de Plantas/análise
15.
BMC Genomics ; 18(1): 775, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29020945

RESUMO

BACKGROUND: The harsh environment on the Qinghai-Tibetan Plateau gives Tibetan hulless barley (Hordeum vulgare var. nudum) great ability to resist adversities such as drought, salinity, and low temperature, and makes it a good subject for the analysis of drought tolerance mechanism. To elucidate the specific gene networks and pathways that contribute to its drought tolerance, and for identifying new candidate genes for breeding purposes, we performed a transcriptomic analysis using two accessions of Tibetan hulless barley, namely Z772 (drought-tolerant) and Z013 (drought-sensitive). RESULTS: There were more up-regulated genes of Z772 than Z013 under both mild (5439-VS-2604) and severe (7203-VS-3359) dehydration treatments. Under mild dehydration stress, the pathways exclusively enriched in drought-tolerance genotype Z772 included Protein processing in endoplasmic reticulum, tricarboxylic acid (TCA) cycle, Wax biosynthesis, and Spliceosome. Under severe dehydration stress, the pathways that were mainly enriched in Z772 included Carbon fixation in photosynthetic organisms, Pyruvate metabolism, Porphyrin and chlorophyll metabolism. The main differentially expressed genes (DEGs) in response to dehydration stress and genes whose expression was different between tolerant and sensitive genotypes were presented in this study, respectively. The candidate genes for drought tolerance were selected based on their expression patterns. CONCLUSIONS: The RNA-Seq data obtained in this study provided an initial overview on global gene expression patterns and networks that related to dehydration shock in Tibetan hulless barley. Furthermore, these data provided pathways and a targeted set of candidate genes that might be essential for deep analyzing the molecular mechanisms of plant tolerance to drought stress.


Assuntos
Secas , Perfilação da Expressão Gênica , Hordeum/genética , Água/metabolismo , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Ontologia Genética , Genótipo , Hordeum/citologia , Hordeum/fisiologia , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética
16.
Plant J ; 91(4): 601-612, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28482117

RESUMO

Exploring genes with impact on yield-related phenotypes is the preceding step to accomplishing crop improvements while facing a growing world population. A genome-wide association scan on leaf blade area (LA) in a worldwide spring barley collection (Hordeum vulgare L.), including 125 two- and 93 six-rowed accessions, identified a gene encoding the homeobox transcription factor, Six-rowed spike 1 (VRS1). VRS1 was previously described as a key domestication gene affecting spike development. Its mutation converts two-rowed (wild-type VRS1, only central fertile spikelets) into six-rowed spikes (mutant vrs1, fully developed fertile central and lateral spikelets). Phenotypic analyses of mutant and wild-type leaves revealed that mutants had an increased leaf width with more longitudinal veins. The observed significant increase of LA and leaf nitrogen (%) during pre-anthesis development in vrs1 mutants also implies a link between wider leaf and grain number, which was validated from the association of vrs1 locus with wider leaf and grain number. Histological and gene expression analyses indicated that VRS1 might influence the size of leaf primordia by affecting cell proliferation of leaf primordial cells. This finding was supported by the transcriptome analysis of mutant and wild-type leaf primordia where in the mutant transcriptional activation of genes related to cell proliferation was detectable. Here we show that VRS1 has an independent role on barley leaf development which might influence the grain number.


Assuntos
Hordeum/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Genes Homeobox , Estudo de Associação Genômica Ampla , Genótipo , Hordeum/citologia , Hordeum/crescimento & desenvolvimento , Mutação , Fenótipo , Filogenia , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética
17.
Plant Cell Environ ; 40(8): 1609-1617, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28425570

RESUMO

Many crops grow well on neutral or weakly acidic soils. The ability of roots to elongate under high-external pH would be advantageous for the survival of plants on alkaline soil. We found that root elongation was promoted in some plant species in alkaline-nutrient solution. Barley, but not tomato, root growth was maintained in pH 8 nutrient solution. Fe and Mn were absorbed well from the pH 8 nutrient solution by both barley and tomato plants, suggesting that the different growth responses of these two species may not be caused by insolubilization of transition metals. The ability of intact barley and tomato plants to acidify external solution was comparable; in both species, this ability decreased in plants exposed to pH 8 nutrient solution for 1 w. Conversely, cell proliferation and elongation in barley root apices were facilitated at pH 8 as shown by microscopy and cell-cycle-related gene-expression data; this was not observed in tomato. We propose that barley adapts to alkaline stress by increasing root development.


Assuntos
Diferenciação Celular , Hordeum/citologia , Hordeum/crescimento & desenvolvimento , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Bioensaio , Proliferação de Células , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Concentração de Íons de Hidrogênio , Solanum lycopersicum/crescimento & desenvolvimento , Meristema/citologia , Meristema/crescimento & desenvolvimento , Raízes de Plantas/genética , Soluções
18.
Methods Mol Biol ; 1588: 209-214, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28417371

RESUMO

Fourier transformed mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls (Acebes et al., Front Plant Sci 5:303, 2014; Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Badhan et al., BioMed Res Int 2015: 562952, 2015; Roach et al., Plant Physiol 156:1351-1363, 2011). The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here, we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. This method has been successfully used to study complex enzyme saccharification processes like rumen digestion to identify recalcitrant moieties in low-quality forage which resist rumen digestion (Badhan et al., BioMed Res Int 2015: 562952, 2015), as well as to characterize cell wall mutant lines or transgenic lines expressing exogenous hydrolases (Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Roach et al., Plant Physiol 156:1351-1363, 2011). The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low cost and nondestructive manner.


Assuntos
Células Vegetais/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ração Animal , Animais , Bovinos , Celulose/análise , Fezes/química , Hordeum/química , Hordeum/citologia , Polissacarídeos/química
19.
Plant Cell Environ ; 40(8): 1392-1408, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28164319

RESUMO

The functional implications of root cortical senescence (RCS) are poorly understood. We tested the hypotheses that RCS in barley (1) reduces the respiration and nutrient content of root tissue; (2) decreases radial water and nutrient transport; and (3) is accompanied by increased suberization to protect the stele. Genetic variation for RCS exists between modern germplasm and landraces. Nitrogen and phosphorus deficiency increased the rate of RCS. Maximal RCS, defined as the disappearance of the entire root cortex, reduced root nitrogen content by 66%, phosphorus content by 63% and respiration by 87% compared with root segments with no RCS. Roots with maximal RCS had 90, 92 and 84% less radial water, nitrate and phosphorus transport, respectively, compared with segments with no RCS. The onset of RCS coincided with 30% greater aliphatic suberin in the endodermis. These results support the hypothesis that RCS reduces root carbon and nutrient costs and may therefore have adaptive significance for soil resource acquisition. By reducing root respiration and nutrient content, RCS could permit greater root growth, soil resource acquisition and resource allocation to other plant processes. RCS merits investigation as a trait for improving the performance of barley, wheat, triticale and rye under edaphic stress.


Assuntos
Hordeum/metabolismo , Nitrogênio/metabolismo , Fósforo/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Água/metabolismo , Transporte Biológico , Respiração Celular , Hordeum/citologia , Lipídeos , Solo
20.
Plant Cell Environ ; 40(1): 11-24, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27155357

RESUMO

Drought is a crucial environmental constraint limiting crop production in many parts of the world. microRNA (miRNA) based gene regulation has been shown to act in several pathways, including crop response to drought stress. Sequence based profiling and computational analysis have revealed hundreds of miRNAs and their potential targets in different plant species under various stress conditions, but few have been biologically verified. In this study, 11 candidate miRNAs were tested for their expression profiles in barley. Differences in accumulation of only four miRNAs (Ath-miR169b, Osa-miR1432, Hv-miRx5 and Hv-miR166b/c) were observed between drought-treated and well-watered barley in four genotypes. miRNA targets were predicted using degradome analysis of two, different genotypes, and genotype-specific target cleavage was observed. Inverse correlation of mature miRNA accumulation with miRNA target transcripts was also genotype dependent under drought treatment. Drought-responsive miRNAs accumulated predominantly in mesophyll tissues. Our results demonstrate genotype-specific miRNA regulation under drought stress and evidence for their role in mediating expression of target genes for abiotic stress response in barley.


Assuntos
Secas , Perfilação da Expressão Gênica , Hordeum/genética , Hordeum/fisiologia , MicroRNAs/genética , Estresse Fisiológico/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genótipo , Hordeum/citologia , MicroRNAs/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
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