RESUMO
The current toolbox of cell wall-directed molecular probes has been pivotal for advancing basic and application-oriented plant carbohydrate research; however, it still exhibits limitations regarding target diversity and specificity. Scarcity of probes targeting intramolecular associations between cell wall polymers particularly hinders our understanding of the cell wall microstructure and affects the development of effective means for its efficient deconstruction for bioconversion. Here we report a detailed characterization of a cellulose-binding DNA aptamer CELAPT MINI using a combination of various in vitro biochemical, biophysical, and molecular biology techniques. Our results show evidence for its high specificity towards long non-substituted ß-(1-4)-glucan chains in both crystalline and amorphous forms. Fluorescent conjugates of CELAPT MINI are applicable as in situ cellulose probes and are well suited for various microscopy techniques, including super-resolution imaging. Compatibility of fluorescent CELAPT MINI variants with immunodetection of cell wall matrix polymers enabled them simultaneously to resolve the fibrillar organization of complex cellulose-enriched pulp material and to quantify the level of cellulose masking by xyloglucan and xylan. Using enzymatically, chemically, or genetically modulated Brachypodium internode sections we showed the diversity in cell wall packing among various cell types and even cell wall microdomains. We showed that xylan is the most prominent, but not the only, cellulose-masking agent in Brachypodium internode tissues. These results collectively highlight the hitherto unexplored potential to expand the cell wall probing toolbox with highly specific and versatile in vitro generated polynucleotide probes.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Brachypodium/citologia , Parede Celular/química , Celulose/metabolismo , Brachypodium/genética , Parede Celular/ultraestrutura , Celulose/análise , Celulose/química , Glucanos/química , Glucanos/metabolismo , Glucose/química , Ligação de Hidrogênio , Lignina/genética , Simulação de Acoplamento Molecular , Imagem Molecular , Reação em Cadeia da Polimerase em Tempo Real , Xilanos/química , Xilanos/metabolismo , beta-Glucanas/químicaRESUMO
Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period ("salt stress") or only after root emergence ("salt shock"). Germination was affected at ≥100 mM and root elongation at ≥75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the "salt stress" but to a lesser extent by "salt shock". Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that "salt stress" increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by "salt stress" in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under "salt shock", the cell cycle genes were optimally expressed at 100 mM NaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt.
Assuntos
Brachypodium/efeitos dos fármacos , Estresse Salino/fisiologia , Cloreto de Sódio/farmacologia , Brachypodium/citologia , Brachypodium/genética , Brachypodium/crescimento & desenvolvimento , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Mitose/efeitos dos fármacos , Mitose/genética , Mucoproteínas/genética , Mucoproteínas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Salinidade , Estresse Salino/genéticaRESUMO
The plant cell wall plays a critical role in signaling responses to environmental and developmental cues, acting as both the sensing interface and regulator of plant cell integrity. Wall-associated kinases (WAKs) are plant receptor-like kinases located at the wall-plasma membrane-cytoplasmic interface and implicated in cell wall integrity sensing. WAKs in Arabidopsis thaliana have been shown to bind pectins in different forms under various conditions, such as oligogalacturonides (OG)s in stress response, and native pectin during cell expansion. The mechanism(s) WAKs use for sensing in grasses, which contain relatively low amounts of pectin, remains unclear. WAK genes from the model monocot plant, Brachypodium distachyon were identified. Expression profiling during early seedling development and in response to sodium salicylate and salt treatment was undertaken to identify WAKs involved in cell expansion and response to external stimuli. The BdWAK2 gene displayed increased expression during cell expansion and stress response, in addition to playing a potential role in the hypersensitive response. In vitro binding assays with various forms of commercial polysaccharides (pectins, xylans, and mixed-linkage glucans) and wall-extracted fractions (pectic/hemicellulosic/cellulosic) from both Arabidopsis and Brachypodium leaf tissues provided new insights into the binding properties of BdWAK2 and other candidate BdWAKs in grasses. The BdWAKs displayed a specificity for the acidic pectins with similar binding characteristics to the AtWAKs.
Assuntos
Brachypodium/citologia , Brachypodium/enzimologia , Parede Celular/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Estresse Fisiológico , Brachypodium/genética , Morte Celular , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismoRESUMO
Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.
Assuntos
Brachypodium/citologia , Brachypodium/metabolismo , Brachypodium/imunologia , Parede Celular/metabolismo , Ciclinas/metabolismo , Desenvolvimento Embrionário/fisiologia , Epitopos/imunologia , Epitopos/metabolismo , Citometria de Fluxo , Glicoproteínas/metabolismo , Mucoproteínas/metabolismo , Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de PlantasRESUMO
Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.
Assuntos
Brachypodium/crescimento & desenvolvimento , Germinação , Plântula/crescimento & desenvolvimento , Brachypodium/citologia , Brachypodium/embriologia , Ciclo Celular , DNA de Plantas/análise , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Plântula/citologia , Plântula/embriologiaRESUMO
All multicellular organisms must properly pattern cell types to generate functional tissues and organs. The organized and predictable cell lineages of the Brachypodium leaf enabled us to characterize the role of the MAPK kinase kinase gene BdYODA1 in regulating asymmetric cell divisions. We find that YODA genes promote normal stomatal spacing patterns in both Arabidopsis and Brachypodium, despite species-specific differences in those patterns. Using lineage tracing and cell fate markers, we show that, unexpectedly, patterning defects in bdyoda1 mutants do not arise from faulty physical asymmetry in cell divisions but rather from improper enforcement of alternative cellular fates after division. These cross-species comparisons allow us to refine our understanding of MAPK activities during plant asymmetric cell divisions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Brachypodium/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Estômatos de Plantas/enzimologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brachypodium/citologia , Brachypodium/genética , MAP Quinase Quinase Quinases/genética , Estômatos de Plantas/citologia , Estômatos de Plantas/genética , Especificidade da EspécieRESUMO
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 AminoácidosRESUMO
Brachypodium distachyon provides a particularly appealing object for molecular cytogenetic analysis due to its compact genome and low repetitive DNA content, as well as low (x = 5) basic number of chromosomes easily identifiable on the basis of their morphometric features. Some of these features, such as genome compactness, are shared by the other members of the genus, thus making them amenable for comparative cytogenetic mapping. Cytogenetic infrastructure established for B. distachyon was initially based on fluorescence in situ hybridization with various tandemly repeated sequences as probes. The molecular cytogenetic studies advanced greatly with the development of B. distachyon large DNA insert genomic libraries. These resources coupled with the access to the fully sequenced genome of B. distachyon enabled chromosome painting in monocots for the first time. This pioneering work was subsequently extended to other Brachypodium species, allowing insight into grass karyotype evolution. In this protocol we describe the methods of making somatic and meiotic chromosome preparations, probe labeling, FISH with BAC clones, a strategy for chromosome barcoding and chromosome painting in B. distachyon, and comparative chromosome painting in the other Brachypodium species.
Assuntos
Brachypodium/citologia , Brachypodium/genética , Coloração Cromossômica/métodos , Cromossomos de Plantas/genética , Citogenética/métodos , Genoma de Planta , Hibridização in Situ Fluorescente/métodos , Cariótipo , Meiose , MitoseRESUMO
While Brachypodium distachyon (Brachypodium) is an emerging model for grasses, no expression atlas or gene coexpression network is available. Such tools are of high importance to provide insights into the function of Brachypodium genes. We present a detailed Brachypodium expression atlas, capturing gene expression in its major organs at different developmental stages. The data were integrated into a large-scale coexpression database ( www.gene2function.de), enabling identification of duplicated pathways and conserved processes across 10 plant species, thus allowing genome-wide inference of gene function. We highlight the importance of the atlas and the platform through the identification of duplicated cell wall modules, and show that a lignin biosynthesis module is conserved across angiosperms. We identified and functionally characterised a putative ferulate 5-hydroxylase gene through overexpression of it in Brachypodium, which resulted in an increase in lignin syringyl units and reduced lignin content of mature stems, and led to improved saccharification of the stem biomass. Our Brachypodium expression atlas thus provides a powerful resource to reveal functionally related genes, which may advance our understanding of important biological processes in grasses.
Assuntos
Brachypodium/citologia , Brachypodium/genética , Parede Celular/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Lignina/metabolismo , Arabidopsis/genética , Bases de Dados Genéticas , Oryza/genética , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Transcriptoma/genéticaRESUMO
Plant border cells are specialized cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localized cell separation which is essential for their release to the environment is little understood. Here I present in situ analysis of Brachypodium distachyon, a model organism for grasses which possess type II primary cell walls poor in pectin content. Results suggest similarity in spatial dynamics of pectic homogalacturonan during dicot and monocot border cell release. Integration of observations from different species leads to the hypothesis that this process most likely does not involve degradation of cell wall material but rather uses unique cell wall structural and compositional means enabling both the rigidity of the root cap as well as detachability of given cells on its surface.
Assuntos
Brachypodium/fisiologia , Parede Celular/fisiologia , Raízes de Plantas/citologia , Brachypodium/citologiaRESUMO
Plants optimize carbon assimilation while limiting water loss by adjusting stomatal aperture. In grasses, a developmental innovation-the addition of subsidiary cells (SCs) flanking two dumbbell-shaped guard cells (GCs)-is linked to improved stomatal physiology. Here, we identify a transcription factor necessary and sufficient for SC formation in the wheat relative Brachypodium distachyon. Unexpectedly, the transcription factor is an ortholog of the stomatal regulator AtMUTE, which defines GC precursor fate in Arabidopsis The novel role of BdMUTE in specifying lateral SCs appears linked to its acquisition of cell-to-cell mobility in Brachypodium Physiological analyses on SC-less plants experimentally support classic hypotheses that SCs permit greater stomatal responsiveness and larger range of pore apertures. Manipulation of SC formation and function in crops, therefore, may be an effective approach to enhance plant performance.
Assuntos
Proteínas de Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Brachypodium/citologia , Brachypodium/fisiologia , Estômatos de Plantas/citologia , Estômatos de Plantas/fisiologia , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Comunicação Celular , Movimento CelularRESUMO
This paper presents ChroTeMo, a tool for chromosome territory modelling, accompanied by ChroTeVi-a chromosome territory visualisation software that uses the data obtained by ChroTeMo. These tools have been developed in order to complement the molecular cytogenetic research of interphase nucleus structure in a model grass Brachypodium distachyon. Although the modelling tool has been initially created for one particular species, it has universal application. The proposed version of ChroTeMo allows for generating a model of chromosome territory distribution in any given plant or animal species after setting the initial, species-specific parameters. ChroTeMo has been developed as a fully probabilistic modeller. Due to this feature, the comparison between the experimental data on the structure of a nucleus and the results obtained from ChroTeMo can indicate whether the distribution of chromosomes inside a nucleus is also fully probabilistic or is subjected to certain non-random patterns. The presented tools have been written in Python, so they are multiplatform, portable and easy to read. Moreover, if necessary they can be further developed by users writing their portions of code. The source code, documentation, and wiki, as well as the issue tracker and the list of related articles that use ChroTeMo and ChroTeVi, are accessible in a public repository at Github under GPL 3.0 license.
Assuntos
Brachypodium/citologia , Cromossomos de Plantas/metabolismo , Modelos Biológicos , Software , Benchmarking , Brachypodium/genética , Nucléolo Celular/metabolismoRESUMO
Programmed cell death can be defined as an organized cellular destruction and can be activated throughout plant development, as a defense response against invading pathogens or during environmental stress. The root hair assay presented herein enables in vivo quantitative measurements of programmed cell death based on the morphological changes of dying root hairs. Application of this novel, simple technique eliminates the need for establishing cell suspension cultures, resulting in a significant reduction in time, cost, and labor input. Here, we present a detailed root hair assay protocol for the dicotyledonous model plant Arabidopsis thaliana, where results from germination to scoring of cell death can be obtained within 7 days. We also suggest and present a panel of cell death inducing treatments which can be used to study abiotic stress- and mycotoxin-induced programmed cell death in the root hair system in Arabidopsis. A root hair assay protocol for the monocotyledonous model species Brachypodium distachyon is also included.
Assuntos
Arabidopsis/citologia , Brachypodium/citologia , Morte Celular , Técnicas Citológicas , Arabidopsis/anatomia & histologia , Brachypodium/anatomia & histologia , Germinação , Raízes de Plantas/fisiologiaRESUMO
Brachypodium distachyon has emerged as a model plant for the improvement of grain crops such as wheat, barley and oats and for understanding basic biological processes to facilitate the development of grasses as superior energy crops. Brachypodium is also the first species of the grass subfamily Pooideae with a sequenced genome. For obtaining a better understanding of the mechanisms controlling male gametophyte development in B. distachyon, here we report the cellular changes during the stages of anther development, with special reference to the development of the anther wall. Brachypodium anthers are tetrasporangiate and follow the typical monocotyledonous-type anther wall formation pattern. Anther differentiation starts with the appearance of archesporial cells, which divide to generate primary parietal and primary sporogenous cells. The primary parietal cells form two secondary parietal layers. Later, the outer secondary parietal layer directly develops into the endothecium and the inner secondary parietal layer forms an outer middle layer and inner tapetum by periclinal division. The anther wall comprises an epidermis, endothecium, middle layer and the secretory-type tapetum. Major documented events of anther development include the degradation of a secretory-type tapetum and middle layer during the course of development and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The tapetum undergoes degeneration at the tetrad stage and disintegrates completely at the bicellular stage of pollen development. The distribution of insoluble polysaccharides in the anther layers and connective tissue through progressive developmental stages suggests their role in the development of male gametophytes. Until sporogenous cell stage, the amount of insoluble polysaccharides in the anther wall was negligible. However, abundant levels of insoluble polysaccharides were observed during microspore mother cell and tetrad stages and gradually declined during the free microspore and vacuolated microspore stages to undetectable level at the mature stage. Thus, the cellular features in the development of anthers in B. distachyon share similarities with anther and pollen development of other members of Poaceae.
Assuntos
Brachypodium/citologia , Pólen/ultraestrutura , Brachypodium/crescimento & desenvolvimento , Flores/ultraestrutura , Microscopia Eletroquímica de VarreduraRESUMO
Polysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5-10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC-MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty-three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ-specific expression consistent with proteomics results could be observed as well as cell-specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls.
Assuntos
Biocombustíveis , Brachypodium/química , Parede Celular/química , Proteínas de Plantas/análise , Proteoma/análise , Brachypodium/citologia , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/química , Imuno-Histoquímica , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Caules de Planta/química , Proteoma/química , Proteoma/metabolismoRESUMO
The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.
Assuntos
Acetilesterase/metabolismo , Arabidopsis/fisiologia , Aspergillus nidulans/enzimologia , Brachypodium/fisiologia , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Acetilação , Acetilesterase/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/imunologia , Ascomicetos/patogenicidade , Aspergillus nidulans/genética , Botrytis/patogenicidade , Brachypodium/citologia , Brachypodium/genética , Brachypodium/imunologia , Resistência à Doença , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Peróxido de Hidrogênio/metabolismo , Pectinas/metabolismo , Componentes Aéreos da Planta , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/patogenicidade , Regulação para Cima , Xanthomonas/patogenicidadeRESUMO
In several dicotyledonous species, NAC transcription factors act as master switches capable of turning on programmes of secondary cell-wall synthesis and cell death. This work used an oestradiol-inducible system to overexpress the NAC transcription factor BdSWN5 in the monocot model Brachypodium distachyon. This resulted in ectopic secondary cell-wall formation in both roots and shoots. Some of the genes upregulated in the process were a secondary cell-wall cellulose synthase (BdCESA4), a xylem-specific protease (BdXCP1) and an orthologue of AtMYB46 (BdMYB1). While activation of BdMYB1 may not be direct, this study showed that BdSWN5 is capable of transactivating the BdXCP1 promoter through two conserved binding sites. In the course of Brachypodium development, the BdXCP1 promoter was observed to be active in all types of differentiating tracheary elements. Together, these results suggest that Brachypodium SWNs can act as switches that turn on secondary cell-wall synthesis and programmed cell death.
Assuntos
Brachypodium/citologia , Brachypodium/metabolismo , Parede Celular/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Brachypodium/genética , Morte Celular , Parede Celular/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Folhas de Planta/citologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Nicotiana/citologia , Nicotiana/genética , Fatores de Transcrição/genética , Ativação Transcricional , Xilema/genéticaRESUMO
The drought-tolerant grass Brachypodium distachyon is an emerging model species for temperate grasses and cereal crops. To explore the usefulness of this species for drought studies, a reproducible in vivo drought assay was developed. Spontaneous soil drying led to a 45% reduction in leaf size, and this was mostly due to a decrease in cell expansion, whereas cell division remained largely unaffected by drought. To investigate the molecular basis of the observed leaf growth reduction, the third Brachypodium leaf was dissected in three zones, namely proliferation, expansion, and mature zones, and subjected to transcriptome analysis, based on a whole-genome tiling array. This approach allowed us to highlight that transcriptome profiles of different developmental leaf zones respond differently to drought. Several genes and functional processes involved in drought tolerance were identified. The transcriptome data suggest an increased energy availability in the proliferation zones, along with an up-regulation of sterol synthesis that may influence membrane fluidity. This information may be used to improve the tolerance of temperate cereals to drought, which is undoubtedly one of the major environmental challenges faced by agriculture today and in the near future.
Assuntos
Brachypodium/crescimento & desenvolvimento , Secas , Folhas de Planta/crescimento & desenvolvimento , Estresse Fisiológico , Brachypodium/citologia , Brachypodium/fisiologia , Folhas de Planta/citologia , Folhas de Planta/fisiologiaRESUMO
Plant responses to drought are regulated by complex genetic and epigenetic networks leading to rapid reprogramming of plant growth. miRNAs have been widely indicated as key players in the regulation of growth and development. The role of miRNAs in drought response was investigated in young leaves of Brachypodium distachyon, a drought-tolerant monocot model species. Adopting an in vivo drought assay, shown to cause a dramatic reduction in leaf size, mostly due to reduced cell expansion, small RNA libraries were produced from proliferating and expanding leaf cells. Next-generation sequencing data were analyzed using an in-house bioinformatics pipeline allowing the identification of 66 annotated miRNA genes and 122 new high confidence predictions greatly expanding the number of known Brachypodium miRNAs. In addition, we identified four TAS3 loci and a large number of siRNA-producing loci that show characteristics suggesting that they may represent young miRNA genes. Most miRNAs showed a high expression level, consistent with their involvement in early leaf development and cell identity. Proliferating and expanding leaf cells respond differently to drought treatment and differential expression analyses suggest novel evidence for an miRNA regulatory network controlling cell division in both normal and stressed conditions and demonstrate that drought triggers a genetic reprogramming of leaf growth in which miRNAs are deeply involved.
Assuntos
Brachypodium/genética , Brachypodium/fisiologia , Secas , MicroRNAs/genética , Folhas de Planta/crescimento & desenvolvimento , RNA de Plantas/genética , Estresse Fisiológico/genética , Sequência de Bases , Brachypodium/citologia , Brachypodium/crescimento & desenvolvimento , Divisão Celular/genética , Sequência Conservada , Loci Gênicos/genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Folhas de Planta/genéticaRESUMO
In most eukaryotes that have been studied, the telomeres cluster into a bouquet early in meiosis, and in wheat and its relatives and in Arabidopsis the centromeres pair at the same time. In Arabidopsis, the telomeres do not cluster as a typical telomere bouquet on the nuclear membrane but are associated with the nucleolus both somatically and at the onset of meiosis. We therefore assessed whether Brachypodium distachyon, a monocot species related to cereals and whose genome is approximately twice the size of Arabidopsis thaliana, also exhibited an atypical telomere bouquet and centromere pairing. In order to investigate the occurrence of a bouquet and centromere pairing in B distachyon, we first had to establish protocols for studying meiosis in this species. This enabled us to visualize chromosome behaviour in meiocytes derived from young B distachyon spikelets in three-dimensions by fluorescent in situ hybridization (FISH), and accurately to stage meiosis based on chromatin morphology in relation to spikelet size and the timing of sample collection. Surprisingly, this study revealed that the centromeres clustered as a single site at the same time as the telomeres also formed a bouquet or single cluster.