RESUMEN
Plant morphogenesis is governed by the mechanics of the cell wall-a stiff and thin polymeric box that encloses the cells. The cell wall is a highly dynamic composite material. New cell walls are added during cell division. As the cells continue to grow, the properties of cell walls are modulated to undergo significant changes in shape and size without breakage. Spatial and temporal variations in cell wall mechanical properties have been observed. However, how they relate to cell division remains an outstanding question. Here, we combine time-lapse imaging with local mechanical measurements via atomic force microscopy to systematically map the cell wall's age and growth, with their stiffness. We make use of two systems, Marchantia polymorpha gemmae, and Arabidopsis thaliana leaves. We first characterize the growth and cell division of M. polymorpha gemmae. We then demonstrate that cell division in M. polymorpha gemmae results in the generation of a temporary stiffer and slower-growing new wall. In contrast, this transient phenomenon is absent in A. thaliana leaves. We provide evidence that this different temporal behavior has a direct impact on the local cell geometry via changes in the junction angle. These results are expected to pave the way for developing more realistic plant morphogenetic models and to advance the study into the impact of cell division on tissue growth.
Asunto(s)
Arabidopsis , Marchantia , Arabidopsis/genética , Marchantia/genética , Hojas de la Planta , Pared Celular , PolímerosRESUMEN
Hemicellulose polysaccharides influence assembly and properties of the plant primary cell wall (PCW), perhaps by interacting with cellulose to affect the deposition and bundling of cellulose fibrils. However, the functional differences between plant cell wall hemicelluloses such as glucomannan, xylan, and xyloglucan (XyG) remain unclear. As the most abundant hemicellulose, XyG is considered important in eudicot PCWs, but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned ß-galactoglucomannan (ß-GGM) is widespread in eudicot PCWs and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of ß-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state nuclear magnetic resonance indicated that ß-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis ß-GGM synthesis mutants show no obvious growth defects, genetic crosses between ß-GGM and XyG mutants produce exacerbated phenotypes compared with XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work opens avenues to study the roles of ß-GGM and XyG in PCWs.
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Arabidopsis , Xilanos , Arabidopsis/genética , Pared Celular/química , CelulosaRESUMEN
In eukaryotes, most RNA molecules are exported into the cytoplasm after transcription. Long noncoding RNAs (lncRNAs) reside and function primarily inside the nucleus, but nuclear localization of mRNAs has been considered rare in both animals and plants. Here we show that Arabidopsis anaphase-promoting complex/cyclosome (APC/C) coactivator genes CDC20 and CCS52B (CDH1 ortholog) are co-expressed with their target cyclin B genes (CYCBs) during mitosis. CYCB transcripts can be exported and translated; however, CDC20 and CCS52B mRNAs are confined to the nucleus at prophase, and the cognate proteins are not translated until the redistribution of the mRNAs to the cytoplasm after nuclear envelope breakdown (NEBD) at prometaphase. The 5' untranslated region (UTR) plays dual roles in CDC20 mRNA nuclear localization and translation. Mitotic accumulation of CDC20 and CCS52B transcripts enables the timely and rapid activation of APC/C, while the nuclear sequestration of these transcripts at prophase appears to protect cyclins from precocious degradation.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular , Núcleo Celular/genética , Tallos de la Planta/metabolismo , ARN Mensajero/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Tallos de la Planta/citología , Tallos de la Planta/genética , ARN Mensajero/genética , Nicho de Células MadreRESUMEN
The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.
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Brachypodium , Xilanos , Animales , Humanos , Xilanos/metabolismo , Lignina/metabolismo , Brachypodium/metabolismo , Pared Celular/metabolismoRESUMEN
BACKGROUND: Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome. RESULTS: We show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell. CONCLUSIONS: Our results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes.
Asunto(s)
Flavonas/análisis , Primulaceae/ultraestructura , Tricomas/ultraestructura , Microscopía , Microscopía Electrónica , Primulaceae/químicaRESUMEN
In addition to transcriptional regulation, gene expression is further modulated through mRNA spatiotemporal distribution, by RNA movement between cells, and by RNA localization within cells. Here, we have adapted RNA fluorescence in situ hybridization (FISH) to explore RNA localization in Arabidopsis (Arabidopsis thaliana). We show that RNA FISH on sectioned material can be applied to investigate the tissue and subcellular localization of meristem and flower development genes, cell cycle transcripts, and plant long noncoding RNAs. We also developed double RNA FISH to dissect the coexpression of different mRNAs at the shoot apex and nuclear-cytoplasmic separation of cell cycle gene transcripts in dividing cells. By coupling RNA FISH with fluorescence immunocytochemistry, we further demonstrate that a gene's mRNA and protein may be simultaneously detected, for example revealing uniform distribution of PIN-FORMED1 (PIN1) mRNA and polar localization of PIN1 protein in the same cells. Therefore, our method enables the visualization of gene expression at both transcriptional and translational levels with subcellular spatial resolution, opening up the possibility of systematically tracking the dynamics of RNA molecules and their cognate proteins in plant cells.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flores/metabolismo , Hibridación Fluorescente in Situ/métodos , ARN Nuclear/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Meristema/genética , Meristema/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , ARN Nuclear/genéticaAsunto(s)
Evolución Biológica , Pared Celular , Pared Celular/ultraestructura , Semillas , Adaptación Fisiológica , FilogeniaRESUMEN
Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3-4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by â¼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell-cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.
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Arabidopsis/crecimiento & desarrollo , Tamaño de la Célula , Regulación de la Expresión Génica de las Plantas , Meristema/crecimiento & desarrollo , Nicho de Células Madre , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo Celular , División Celular , Membrana Celular/metabolismo , Replicación del ADN , Genes de Plantas , Homeostasis , Proteínas Luminiscentes/metabolismo , Distribución Normal , Brotes de la Planta/crecimiento & desarrolloRESUMEN
BACKGROUND: Cell culture methods allow the detailed observations of individual plant cells and their internal processes. Whereas cultured cells are more amenable to microscopy, they have had limited use when studying the complex interactions between cell populations and responses to external signals associated with tissue and whole plant development. Such interactions result in the diverse range of cell shapes observed in planta compared to the simple polygonal or ovoid shapes in vitro. Microfluidic devices can isolate the dynamics of single plant cells but have restricted use for providing a tissue-like and fibrous extracellular environment for cells to interact. A gap exists, therefore, in the understanding of spatiotemporal interactions of single plant cells interacting with their three-dimensional (3D) environment. A model system is needed to bridge this gap. For this purpose we have borrowed a tool, a 3D nano- and microfibre tissue scaffold, recently used in biomedical engineering of animal and human tissue physiology and pathophysiology in vitro. RESULTS: We have developed a method of 3D cell culture for plants, which mimics the plant tissue environment, using biocompatible scaffolds similar to those used in mammalian tissue engineering. The scaffolds provide both developmental cues and structural stability to isolated callus-derived cells grown in liquid culture. The protocol is rapid, compared to the growth and preparation of whole plants for microscopy, and provides detailed subcellular information on cells interacting with their local environment. We observe cell shapes never observed for individual cultured cells. Rather than exhibiting only spheroid or ellipsoidal shapes, the cells adapt their shape to fit the local space and are capable of growing past each other, taking on growth and morphological characteristics with greater complexity than observed even in whole plants. Confocal imaging of transgenic Arabidopsis thaliana lines containing fluorescent microtubule and actin reporters enables further study of the effects of interactions and complex morphologies upon cytoskeletal organisation both in 3D and in time (4D). CONCLUSIONS: The 3D culture within the fibre scaffolds permits cells to grow freely within a matrix containing both large and small spaces, a technique that is expected to add to current lithographic technologies, where growth is carefully controlled and constricted. The cells, once seeded in the scaffolds, can adopt a variety of morphologies, demonstrating that they do not need to be part of a tightly packed tissue to form complex shapes. This points to a role of the immediate nano- and micro-topography in plant cell morphogenesis. This work defines a new suite of techniques for exploring cell-environment interactions.
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Arabidopsis/crecimiento & desarrollo , Técnicas de Cultivo de Célula/métodos , Morfogénesis , Células Vegetales/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Células Cultivadas , Andamios del TejidoRESUMEN
Movement of cellulose synthase particles have so far been observed on the plant epidermis that are amenable to confocal imaging, yielding appreciable signal and resolution to observe small plasma membrane-localised particles. Presented here is a method, using airyscan confocal microscopy, that permits similar information to be obtained at depth within the developing protoxylem vessels of intact roots.
RESUMEN
Scots pine (Pinus sylvestris L.) is an evergreen coniferous tree with wide distribution and good growth performance in a range of habitats. Therefore, wood from P. sylvestris is produced in many managed forests and is frequently used in industry. Despite the importance of pine wood, we still do not fully understand its molecular structure what limits improvements in its processing. One of the basic features leading to variation in wood properties is the presence of earlywood and latewood which form annual growth rings. Here, we characterise biochemical traits that differentiate cell walls of earlywood and latewood in Scots pine. We discover that latewood is less recalcitrant to enzymatic digestion, with galactoglucomannan showing particularly pronounced difference in accessibility. Interestingly, characterisation of lignin reveals a higher proportion of coniferaldehydes in pine latewood and suggests the presence of a different linkage landscape in this wood type. With complementary analysis of wood polysaccharides this enabled us to propose the first detailed molecular model of earlywood and latewood and to conclude that the variation in lignin structure is likely the main determinant of differences in recalcitrance observed between the two wood types in pine. Our discoveries lay the foundation for improvements in industrial processes that use pine wood since we show clear pathways for increasing the efficiency of enzymatic processing of this renewable material. Our work will help guide future breeding of pine trees with desired timber properties and can help link molecular structure of softwood cell walls to function of the different types of xylem in conifers.
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Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.
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Lignina , Madera , Biomasa , CelulosaRESUMEN
There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Pared Celular/química , Xilanos/biosíntesis , Xilema/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Pared Celular/ultraestructura , Regulación de la Expresión Génica de las Plantas , Aparato de Golgi/metabolismo , Mutagénesis Insercional , Mutación , Pentosiltransferasa/metabolismo , Filogenia , Xilema/ultraestructura , Xilosa/metabolismo , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Many research questions require the study of plant morphology, in particular cells and tissues, as close to their native context as possible and without physical deformations from some preparatory chemical reagents or sample drying. Cryo-scanning electron microscopy (cryoSEM) involves rapid freezing and maintenance of the sample at an ultra-low temperature for detailed surface imaging by a scanning electron beam. The data are useful for exploring tissue/cell morphogenesis, plus an additional cryofracture/cryoplaning/milling step gives information on air and water spaces as well as subcellular ultrastructure. This review gives an overview from sample preparation through to imaging and a detailed account of how this has been applied across diverse areas of plant research. Future directions and improvements to the technique are discussed.
RESUMEN
Plant cell walls are composites of various carbohydrates, proteins and other compounds. Cell walls provide plants with strength and protection, and also represent the most abundant source of renewable biomass. Despite the importance of plant cell walls, comparatively little is known about the identities of genes and functions of proteins involved in their biosynthesis. The model plant Arabidopsis and the availability of its genome sequence have been invaluable for the identification and functional characterization of genes encoding enzymes involved in plant cell-wall biosynthesis. This review covers recent progress in the identification and characterization of genes encoding proteins involved in the biosynthesis of Arabidopsis cell-wall polysaccharides and arabinogalactan proteins. These studies have improved our understanding of both the mechanisms of cell-wall biosynthesis and the functions of various cell-wall polymers, and have highlighted areas where further research is needed.
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Arabidopsis/genética , Pared Celular/enzimología , Mucoproteínas/biosíntesis , Polisacáridos/biosíntesis , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/genética , Celulosa/biosíntesis , Genes de Plantas , Glucanos/biosíntesis , Mananos/biosíntesis , Pectinas/biosíntesis , Proteínas de Plantas/biosíntesis , Xilanos/biosíntesisRESUMEN
A rapidly increasing body of literature suggests that many biological processes are driven by phase separation within polymer mixtures. Liquid-liquid phase separation can lead to the formation of membrane-less organelles, which are thought to play a wide variety of roles in cell metabolism, gene regulation or signaling. One of the characteristics of these systems is that they are poised at phase transition boundaries, which makes them perfectly suited to elicit robust cellular responses to often very small changes in the cell's "environment". Recent observations suggest that, also in the semi-solid environment of plant cell walls, phase separation not only plays a role in wall patterning, hydration and stress relaxation during growth, but also may provide a driving force for cell wall expansion. In this context, pectins, the major polyanionic polysaccharides in the walls of growing cells, appear to play a critical role. Here, we will discuss (i) our current understanding of the structure-function relationship of pectins, (ii) in vivo evidence that pectin modification can drive critical phase transitions in the cell wall, (iii) how such phase transitions may drive cell wall expansion in addition to turgor pressure and (iv) the periodic cellular processes that may control phase transitions underlying cell wall assembly and expansion.
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Many species have cuticular striations that play a range of roles, from pollinator attraction to surface wettability. In Hibiscus trionum, the striations span multiple cells at the base of the petal to form a pattern that produces a type of iridescence. It is postulated, using theoretical models, that the pattern of striations could result from mechanical instabilities. By combining the application of mechanical stress with high-resolution imaging, we demonstrate that the cuticle buckles to create a striated pattern. Through mechanical modeling and cryo-SEM fractures, we show that the cuticle behaves like a bilayer system with a stiff film on a compliant substrate. The pattern of buckling aligns with the direction of the stress to create a larger-scale pattern. Our findings contribute to the understanding of the formation of tissue-wide patterns in living organisms.
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Hibiscus/química , Luz , Fenómenos Mecánicos/efectos de la radiación , Fuerza Compresiva , Microscopía por Crioelectrón , Flores/química , Flores/efectos de la radiación , Flores/ultraestructura , Hibiscus/crecimiento & desarrollo , Hibiscus/efectos de la radiación , Modelos Teóricos , Semillas/química , Semillas/crecimiento & desarrollo , Estrés MecánicoRESUMEN
Mitogens trigger cell division in animals. In plants, cytokinins, a group of phytohormones derived from adenine, stimulate cell proliferation. Cytokinin signaling is initiated by membrane-associated histidine kinase receptors and transduced through a phosphorelay system. We show that in the Arabidopsis shoot apical meristem (SAM), cytokinin regulates cell division by promoting nuclear shuttling of Myb-domain protein 3R4 (MYB3R4), a transcription factor that activates mitotic gene expression. Newly synthesized MYB3R4 protein resides predominantly in the cytoplasm. At the G2-to-M transition, rapid nuclear accumulation of MYB3R4-consistent with an associated transient peak in cytokinin concentration-feeds a positive feedback loop involving importins and initiates a transcriptional cascade that drives mitosis and cytokinesis. An engineered nuclear-restricted MYB3R4 mimics the cytokinin effects of enhanced cell proliferation and meristem growth.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , División Celular , Citocininas/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Puntos de Control del Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Carioferinas/metabolismo , Meristema/metabolismo , Mitosis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Transactivadores/genéticaRESUMEN
We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.
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Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Flores/crecimiento & desarrollo , Flores/genética , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Flores/anatomía & histología , Flores/citología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes de Plantas , Morfogénesis/genética , Mutación/genéticaRESUMEN
The potential for using cellulosic biomass as a source of fuel has renewed interest into how the large cellulose synthase complex deposits cellulose within the woody secondary walls of plants. This complex sits within the plasma membrane where it synthesizes numerous glucan chains which bond together to form the strong cellulose microfibril. The maintenance and guidance of the complex at the plasma membrane and its delivery to sites of secondary wall formation require the involvement of the cytoskeleton. In the present paper, we discuss the dynamics of the complex at the cell cortex and what is known about its assembly and trafficking.