RESUMEN
The plant cytokinetic microtubule array, called the phragmoplast, exhibits higher microtubule dynamics in its center (midzone) than at the periphery (distal zone). This behavior is known as the axial asymmetry. Despite being a major characteristic of the phragmoplast, little is known about regulators of this phenomenon. Here we address the role of microtubule nucleation in axial asymmetry by characterizing MACERATOR (MACET) proteins in Arabidopsis thaliana and Nicotiana benthamiana with a combination of genetic, biochemical, and live-cell imaging assays, using photo-convertible microtubule probes, and modeling. MACET paralogs accumulate at the shrinking microtubule ends and decrease the tubulin OFF rate. Loss of MACET4 and MACET5 function abrogates axial asymmetry by suppressing microtubule dynamicity in the midzone. MACET4 also narrows the microtubule nucleation angle at the phragmoplast leading edge and functions as a microtubule tethering factor for AUGMIN COMPLEX SUBUNIT 7 (AUG7). The macet4 macet5 double mutant shows diminished clustering of AUG7 in the phragmoplast distal zone. Knockout of AUG7 does not affect MACET4 localization, axial asymmetry, or microtubule nucleation angle, but increases phragmoplast length and slows down phragmoplast expansion. The mce4-1 mce5 aug7-1 triple knockout is not viable. Experimental data and modeling demonstrate that microtubule nucleation factors regulate phragmoplast architecture and axial asymmetry directly by generating new microtubules and indirectly by modulating the abundance of free tubulin.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nicotiana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
The metabolism of tetrahydrofolate (H4PteGlun)-bound one-carbon (C1) units (C1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C1 units in specific organelles and tissues. One possible source of C1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H4PteGlun) from formate and tetrahydrofolate (H4PteGlun). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower Km and kcat values with pentaglutamyl tetrahydrofolate (H4PteGlu5) as compared to monoglutamyl tetrahydrofolate (H4PteGlu1), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H4PteGlun + 5,10-methylene-H4PteGlun and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C1 network in roots with C1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H4PteGlun, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.
RESUMEN
Organelles function as hubs of cellular metabolism and elements of cellular architecture. In addition to 3 spatial dimensions that describe the morphology and localization of each organelle, the time dimension describes complexity of the organelle life cycle, comprising formation, maturation, functioning, decay, and degradation. Thus, structurally identical organelles could be biochemically different. All organelles present in a biological system at a given moment of time constitute the organellome. The homeostasis of the organellome is maintained by complex feedback and feedforward interactions between cellular chemical reactions and by the energy demands. Synchronized changes of organelle structure, activity, and abundance in response to environmental cues generate the fourth dimension of plant polarity. Temporal variability of the organellome highlights the importance of organellomic parameters for understanding plant phenotypic plasticity and environmental resiliency. Organellomics involves experimental approaches for characterizing structural diversity and quantifying the abundance of organelles in individual cells, tissues, or organs. Expanding the arsenal of appropriate organellomics tools and determining parameters of the organellome complexity would complement existing -omics approaches in comprehending the phenomenon of plant polarity. To highlight the importance of the fourth dimension, this review provides examples of organellome plasticity during different developmental or environmental situations.
Asunto(s)
Orgánulos , Plantas , Orgánulos/metabolismoAsunto(s)
Caspasas/clasificación , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/clasificación , Proteínas de Plantas/clasificación , Terminología como Asunto , Animales , Caspasas/química , Caspasas/metabolismo , Consenso , Humanos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.
Asunto(s)
Arabidopsis/genética , Citocinesis/genética , Forminas/metabolismo , Nicotiana/genética , Tionas/farmacología , Uracilo/análogos & derivados , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Citocinesis/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Forminas/genética , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/fisiología , Tubulina (Proteína)/metabolismo , Uracilo/farmacologíaRESUMEN
BACKGROUND: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. RESULTS: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40-55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. CONCLUSIONS: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.
Asunto(s)
Apoptosis , Animales , Caspasas , Muerte Celular , Necrosis , Células Vegetales , PlantasRESUMEN
Here, we show that the embryophyte (land-plant)-specific protein MACERATOR4 (MACET4) binds microtubules in vitro and in vivo, promotes microtubule polymerization at sub-critical tubulin concentrations, decreases the lag phase in microtubule bulk polymerization assays, and colocalizes with microtubule nucleation sites. Furthermore, we find that MACET4 forms oligomers that induce aster formation in vitro in a manner that is similar to aster formation mediated by centrosomes and TPX2. MACET4 is expressed during cell division and accumulates at the microtubule nucleation regions of the plant-specific cytokinetic microtubule array, the phragmoplast. We found that MACET4 localizes to the preprophase band and the cortical division zone, but not the spindle. MACET4 appears as cytoplasmic foci in vivo and forms octamers in vitro Transient expression in tobacco leaf pavement cells results in labeling of shrinking plus- and minus-ends. MACET4 facilitates microtubule depolymerization by increasing the frequency of catastrophes in vivo and by suppressing rescues in vitro Microtubules formed in the presence of MACET4 in vitro are shorter, most likely due to the depletion of the free tubulin pool. Accordingly, MACET4 knockdown results in longer phragmoplasts. We conclude that the direct activity of MACET4 is in promoting microtubule nucleation.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Nicotiana/metabolismo , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Centrosoma/metabolismo , Huso Acromático/metabolismo , Nicotiana/genéticaRESUMEN
The epidermis is hypothesized to play a signalling role during plant development. One class of mutants showing defects in signal transduction and radial patterning are those in sterol biosynthesis. The expectation is that living cells require sterols, but it is not clear that all cell types express sterol biosynthesis genes. The HYDRA1 (HYD1) gene of Arabidopsis encodes sterol Δ8-Δ7 isomerase, and although hyd1 seedlings are defective in radial patterning across several tissues, we show that the HYD1 gene is expressed most strongly in the root epidermis. Transgenic activation of HYD1 transcription in the epidermis of hyd1 null mutants reveals a major role in root patterning and growth. HYD1 expression in the vascular tissues and root meristem, though not endodermis or pericycle, also leads to some phenotypic rescue. Phenotypic rescue is associated with rescued patterning of the PIN1 and PIN2 auxin efflux carriers. The importance of the epidermis in controlling root growth and development is proposed to be, in part, due to its role as a site for sterol biosynthesis, and auxin is a candidate for the non-cell-autonomous signal.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Meristema/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Esteroide Isomerasas/metabolismo , Esteroles/metabolismo , Arabidopsis/embriología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Meristema/embriología , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Plantones/embriología , Plantones/genética , Transducción de Señal/genética , Esteroide Isomerasas/genética , Activación Transcripcional/genéticaRESUMEN
The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development. The establishment of auxin gradients requires the asymmetric distribution of members of the auxin efflux carrier PIN-FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localisation. N-Ethylmaleimide-sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q- or R-SNAREs based on their sequence. We analysed gain- and loss-of-function mutants, dominant-negative transgenics and localisation of the Arabidopsis R-SNARE VAMP714 protein to understand its function. We demonstrate that VAMP714 is essential for the insertion of PINs into the plasma membrane, for polar auxin transport, root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co-localises with endoplasmic reticulum and Golgi vesicles and with PIN proteins at the plasma membrane. It is proposed that VAMP714 mediates the delivery of PIN-carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714-dependent PIN/auxin transport system to control development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Ácidos Indolacéticos , Raíces de Plantas/metabolismo , Proteínas SNARERESUMEN
TPX2 proteins were first identified in vertebrates as a key mitotic spindle assembly factor. Subsequent studies demonstrated that TPX2 is an intricate protein, with functionally and structurally distinct domains and motifs including Aurora kinase-binding, importin-binding, central microtubule-binding, and C-terminal TPX2 conserved domain, among others. The first plant TPX2-like protein, WAVE-DAMPENED2, was identified in Arabidopsis as a dominant mutation responsible for reducing the waviness of roots grown on slanted agar plates. Each plant genome encodes at least one 'canonical' protein with all TPX2 domains and a family of proteins (20 in Arabidopsis) that diversified to contain only some of the domains. Although all plant TPX2-family proteins to date bind microtubules, they function in distinct processes such as cell division, regulation of hypocotyl cell elongation by hormones and light signals, vascular development, or abiotic stress tolerance. Consequently, their expression patterns, regulation, and functions have diverged considerably. Here we summarize the current body of knowledge surrounding plant TPX2-family proteins.
Asunto(s)
Arabidopsis , Proteínas Asociadas a Microtúbulos , Proteínas de Plantas/genética , Arabidopsis/genética , Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos , PeroxidasasRESUMEN
Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and ß-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and ß-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.
Asunto(s)
Resistencia a la Enfermedad/inmunología , Peroxisomas/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Potyviridae , Triticum/inmunología , Triticum/virología , Quitinasas/metabolismo , Clorofila/metabolismo , Glucano 1,3-beta-Glucosidasa/metabolismo , Oxidación-Reducción , Peroxidasas/metabolismo , Peroxisomas/genética , Peroxisomas/virología , Fenotipo , Hojas de la Planta/inmunología , Hojas de la Planta/virología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Although peroxisomes play a key role in plant metabolism under both normal and stressful growth conditions, the impact of drought and heat stress on the peroxisomes remains unknown. Quinoa represents an informative system for dissecting the impact of abiotic stress on peroxisome proliferation because it is adapted to marginal environments. Here we determined the correlation of peroxisome abundance with physiological responses and yield under heat, drought and heat plus drought stresses in eight genotypes of quinoa. We found that all stresses caused a reduction in stomatal conductance and yield. Furthermore, H2 O2 content increased under drought and heat plus drought. Principal component analysis demonstrated that peroxisome abundance correlated positively with H2 O2 content in leaves and correlated negatively with yield. Pearson correlation coefficient for yield and peroxisome abundance (r = -0.59) was higher than for commonly used photosynthetic efficiency (r = 0.23), but comparable to those for classical stress indicators such as soil moisture content (r = 0.51) or stomatal conductance (r = 0.62). Our work established peroxisome abundance as a cellular sensor for measuring responses to heat and drought stress in the genetically diverse populations. As heat waves threaten agricultural productivity in arid climates, our findings will facilitate identification of genetic markers for improving yield of crops under extreme weather patterns.
Asunto(s)
Chenopodium quinoa/genética , Chenopodium quinoa/metabolismo , Respuesta al Choque Térmico/fisiología , Peroxisomas/metabolismo , Productos Agrícolas/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Calor , Peróxido de Hidrógeno/metabolismo , Peroxisomas/genética , Fotosíntesis/genética , Fotosíntesis/fisiología , Filogenia , Estomas de Plantas/metabolismoRESUMEN
Plant morphogenesis relies on the accurate positioning of the partition (cell plate) between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which consists of microtubules, actin filaments, membrane compartments and associated proteins. The phragmoplast forms between daughter nuclei during the transition from anaphase to telophase. As cells are commonly larger than the originally formed phragmoplast, the construction of the cell plate requires phragmoplast expansion. This expansion depends on microtubule polymerization at the phragmoplast forefront (leading zone) and loss at the back (lagging zone). Leading and lagging zones sandwich the 'transition' zone. A population of stable microtubules in the transition zone facilitates transport of building materials to the midzone where the cell plate assembly takes place. Whereas microtubules undergo dynamic instability in all zones, the overall balance appears to be shifted towards depolymerization in the lagging zone. Polymerization of microtubules behind the lagging zone has not been reported to date, suggesting that microtubule loss there is irreversible. In this Review, we discuss: (1) the regulation of microtubule dynamics in the phragmoplast zones during expansion; (2) mechanisms of the midzone establishment and initiation of cell plate biogenesis; and (3) signaling in the phragmoplast.
Asunto(s)
Citocinesis , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Polimerizacion , Transducción de SeñalRESUMEN
Pits are regions in the cell walls of plant tracheary elements that lack secondary walls. Each pit consists of a space within the secondary wall called a pit chamber, and a modified primary wall called the pit membrane. The pit membrane facilitates transport of solutions between vessel cells and restricts embolisms during drought. Here we analyzed the role of an angiosperm-specific TPX2-like microtubule protein MAP20 in pit formation using Brachypodium distachyon as a model system. Live cell imaging was used to analyze the interaction of MAP20 with microtubules and the impact of MAP20 on microtubule dynamics. MAP20-specific antibody was used to study expression and localization of MAP20 in different cell types during vascular bundle development. We used an artificial microRNAs (amiRNA) knockdown approach to determine the function of MAP20. MAP20 is expressed during the late stages of vascular bundle development and localizes around forming pits and under secondary cell wall thickenings in metaxylem cells. MAP20 suppresses microtubule depolymerization; however, unlike the animal TPX2 counterpart, MAP20 does not cooperate with the γ-tubulin ring complex in microtubule nucleation. Knockdown of MAP20 causes bigger pits, thinner pit membranes, perturbed vasculature development, lower reproductive potential and higher drought susceptibility. We conclude that MAP20 may contribute to drought adaptation by modulating pit size and pit membrane thickness in metaxylem.
Asunto(s)
Brachypodium , Proteínas de Microtúbulos , Proteínas de Plantas , Brachypodium/genética , Brachypodium/fisiología , Pared Celular , Sequías , MicrotúbulosRESUMEN
Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress.
Asunto(s)
Arabidopsis/metabolismo , Gránulos Citoplasmáticos/metabolismo , Nucleasa Microcócica/metabolismo , Procesamiento Postranscripcional del ARN , Estrés Fisiológico , Adaptación Fisiológica , Proteínas de Arabidopsis/metabolismo , Respuesta al Choque Térmico , Cinética , Meristema/citología , Meristema/metabolismo , Nucleasa Microcócica/química , Microtúbulos/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Plant morphogenesis depends on the synchronized anisotropic expansion of individual cells in response to developmental and environmental cues. The magnitude of cell expansion depends on the biomechanical properties of the cell wall, which in turn depends on both its biosynthesis and extensibility. Although the control of cell expansion by the phytohormone auxin is well established, its regulation of cell wall composition, trafficking of H+-ATPases, and K+ influx that drives growth is still being elucidated. Furthermore, the maintenance of auxin fluxes via the interaction between the cytoskeleton and PIN protein recycling on the plasma membrane remains under investigation. This review proposes a model that describes how the cell wall, auxin, microtubule binding-protein CLASP and Kin7/separase complexes, and vesicle trafficking are co-ordinated on a cellular level to mediate cell wall loosening during cell expansion.
Asunto(s)
Pared Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Microtúbulos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Transporte de Proteínas , Separasa/genética , Separasa/metabolismoRESUMEN
The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.
Asunto(s)
Anafase , Picea/citología , Picea/enzimología , Proteínas de Plantas/metabolismo , Semillas/citología , Semillas/enzimología , Separasa/metabolismo , Secuencia de Aminoácidos , Anisotropía , Proliferación Celular , Cromosomas de las Plantas/genética , Clonación Molecular , Citocinesis , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Microtúbulos/metabolismo , Filogenia , Picea/embriología , Transporte de Proteínas , Semillas/embriología , Análisis de Secuencia de ProteínaRESUMEN
Vesicle trafficking plays an important role in cell division, establishment of cell polarity, and translation of environmental cues to developmental responses. However, the molecular mechanisms regulating vesicle trafficking remain poorly understood. Here, we report that the evolutionarily conserved caspase-related protease separase (extra spindle poles [ESP]) is required for the establishment of cell polarity and cytokinesis in Arabidopsis thaliana. At the cellular level, separase colocalizes with microtubules and RabA2a (for RAS genes from rat brainA2a) GTPase-positive structures. Separase facilitates polar targeting of the auxin efflux carrier PIN-formed2 (PIN2) to the rootward side of the root cortex cells. Plants with the radially swollen4 (rsw4) allele with compromised separase activity, in addition to mitotic failure, display isotropic cell growth, perturbation of auxin gradient formation, slower gravitropic response in roots, and cytokinetic failure. Measurements of the dynamics of vesicle markers on the cell plate revealed an overall reduction of the delivery rates of KNOLLE and RabA2a GTPase in separase-deficient roots. Furthermore, dissociation of the clathrin light chain, a protein that plays major role in the formation of coated vesicles, was slower in rsw4 than in the control. Our results demonstrate that separase is a key regulator of vesicle trafficking, which is indispensable for cytokinesis and the establishment of cell polarity.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Polaridad Celular/genética , Citocinesis/genética , Separasa/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microtúbulos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Separasa/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Somatic embryogenesis (SE) is a process of differentiation of cells into a plant bypassing the fusion of gametes. As such, it represents a very powerful tool in biotechnology for propagation of species with a long reproductive cycle or low seed set and production of genetically modified plants with improved traits. SE is also a versatile model to study cellular and molecular mechanisms of plant embryo patterning. The morphology and molecular regulation of SE resemble those of zygotic embryogenesis and begin with establishment of apical-basal asymmetry. The apical domain, the embryo proper, proliferates and eventually gives rise to the plantlet, while the basal part, the embryo suspensor, is terminally differentiated and gradually removed via vacuolar programmed cell death (PCD). This PCD is essential for normal development of the apical domain. Emerging evidence demonstrates that signalling events in the apical and basal domains share homologous components. Here we provide an overview of the main pathways controlling the life and death events during SE.
Asunto(s)
Apoptosis , Fenómenos Fisiológicos de las Plantas , Técnicas de Embriogénesis Somática de Plantas , Plantas/embriología , Proliferación Celular , Desarrollo de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismoRESUMEN
Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY-2 cells treated with 50 µM CdSO4. The cadmium-induced alterations in cell morphology occurred gradually over a period of 3-4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium-induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.