RESUMEN
Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.
RESUMEN
Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Separación de Fases , Pared Celular/metabolismo , Microtúbulos/metabolismo , Xilema/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.
Asunto(s)
Arabidopsis , Arabidopsis/metabolismo , Pared Celular/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Xilema/metabolismoRESUMEN
Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.
Asunto(s)
Áfidos , Arabidopsis , Animales , Arabidopsis/genética , Insectos/genética , Áfidos/genética , Transcriptoma , Tumores de Planta/genéticaRESUMEN
Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.
Asunto(s)
Pared Celular , Xilema , Membrana Celular/metabolismo , Pared Celular/metabolismo , Microtúbulos/metabolismo , Plantas/metabolismo , Agua/metabolismo , Xilema/metabolismoRESUMEN
Phragmoplasts are plant-specific microtubule structures that form cell plates at the cell division plane. During late anaphase, phragmoplasts emerge between daughter nuclei as the derivative of spindle microtubules, and centrifugally expand toward the cell cortex to build cell plates during telophase. Phragmoplasts are composed of short antiparallel microtubules decorated with various microtubule-associated proteins. Mutants of these microtubule-associated proteins exhibit defects in phragmoplast morphology. Quantification of phragmoplast morphology is indispensable for assessing the phenotypes of these mutants. Here, we describe a method to quantify the width of phragmoplasts.
Asunto(s)
Microtúbulos , Anafase , Citocinesis , Proteínas Asociadas a Microtúbulos/genética , MitosisRESUMEN
In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Floema/citología , Floema/crecimiento & desarrollo , Raíces de Plantas/citología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Diferenciación Celular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Meristema/citología , Floema/genética , Floema/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Factores de Transcripción/genética , TranscriptomaRESUMEN
In Arabidopsis thaliana, the vacuolar proton-pumping pyrophosphatase (H+-PPase) is highly expressed in young tissues, which consume large amounts of energy in the form of nucleoside triphosphates and produce pyrophosphate (PPi) as a byproduct. We reported that excess PPi in the H+-PPase loss-of-function fugu5 mutant severely compromised gluconeogenesis from seed storage lipids, arrested cell division in cotyledonary palisade tissue, and triggered compensated cell enlargement; this phenotype was recovered upon sucrose supply. Thus, we provided evidence that the hydrolysis of inhibitory PPi, rather than vacuolar acidification, is the major contribution of H+-PPase during seedling establishment. Here, examination of the epidermis revealed that fugu5 pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from fugu5 background by the yeast cytosolic PPase IPP1, in fugu5-1 AVP1pro::IPP1 transgenic lines, restored the phenotypic aberrations of fugu5 pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis (pck1-2) or the glyoxylate cycle (icl-2; mls-2) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of fugu5 epidermal phenotype. fugu5 had oblong cotyledons similar to those of angustifolia-1 (an-1), whose leaf pavement cells display an abnormal arrangement of cortical microtubules (MTs). To gain insight into the genetic interaction between ANGUSTIFOLIA and H+-PPase in pavement cell differentiation, an-1 fugu5-1 was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, an-1 fugu5-1 pavement cells showed a striking three-dimensional growth phenotype on both abaxial and adaxial sides of cotyledons, which was recovered by hydrolysis of PPi in an-1 fugu5-1 AVP1pro::IPP1. Live imaging revealed that cortical MTs exhibited a reduced velocity, were slightly fragmented and sparse in the above lines compared to the WT. Consistently, addition of PPi in vitro led to a dose-dependent delay of tubulin polymerization, thus supporting a link between PPi and MT dynamics. Moreover, mathematical simulation of three-dimensional growth based on cotyledon proximo-distal and medio-lateral phenotypic quantification implicated restricted cotyledon expansion along the medio-lateral axis in the crinkled surface of an-1 fugu5-1. Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.
RESUMEN
Cytokinesis is fundamental for cell proliferation [1, 2]. In plants, a bipolar short-microtubule array forms the phragmoplast, which mediates vesicle transport to the midzone and guides the formation of cell walls that separate the mother cell into two daughter cells [2]. The phragmoplast centrifugally expands toward the cell cortex to guide cell-plate formation at the cortical division site [3, 4]. Several proteins in the phragmoplast midzone facilitate the anti-parallel bundling of microtubules and vesicle accumulation [5]. However, the mechanisms by which short microtubules are maintained during phragmoplast development, in particular, the behavior of microtubules at the distal zone of phragmoplasts, are poorly understood. Here, we show that a plant-specific protein, CORTICAL MICROTUBULE DISORDERING 4 (CORD4), tethers the conserved microtubule-severing protein katanin to facilitate formation of the short-microtubule array in phragmoplasts. CORD4 was specifically expressed during mitosis and localized to preprophase bands and phragmoplast microtubules. Custom-made two-photon spinning disk confocal microscopy revealed that CORD4 rapidly localized to microtubules in the distal phragmoplast zone during phragmoplast assembly at late anaphase and persisted throughout phragmoplast expansion. Loss of CORD4 caused abnormally long and oblique phragmoplast microtubules and slow expansion of phragmoplasts. The p60 katanin subunit, KTN1, localized to the distal phragmoplast zone in a CORD4-dependent manner. These results suggest that CORD4 tethers KTN1 at phragmoplasts to modulate microtubule length, thereby accelerating phragmoplast growth. This reveals the presence of a distinct machinery to accelerate cytokinesis by regulating the action of katanin.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Citocinesis/genética , Expresión Génica , Katanina/genética , Proteínas Asociadas a Microtúbulos/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Perfilación de la Expresión Génica , Katanina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
An in vitro induction system for xylem vessel formation is a useful tool for visualizing the differentiation of xylem vessel cells. A procedure for inducing xylem vessel cell differentiation in hypocotyls of Arabidopsis thaliana is described here. Metaxylem vessel elements form ectopically in excised hypocotyl tissue following treatment with bikinin. This enables high-resolution imaging of living metaxylem vessel cells. The wide range of resources available for Arabidopsis allows for the visualization of diverse cellular structures, including microtubules and secondary cell walls, in different genetic backgrounds. Use of this system will contribute to the further understanding of the processes by which xylem vessel elements form.
Asunto(s)
Arabidopsis/ultraestructura , Hipocótilo/ultraestructura , Microscopía Fluorescente/métodos , Microscopía de Interferencia/métodos , Xilema/ultraestructura , Arabidopsis/citología , Diferenciación Celular , Pared Celular/ultraestructura , Hipocótilo/citología , Microscopía Confocal/métodos , Microtúbulos , Imagen Óptica/métodos , Coloración y Etiquetado/métodos , Xilema/citologíaRESUMEN
Patterned cell wall deposition is crucial for cell shapes and functions. In Arabidopsis xylem vessels, ROP11 GTPase locally inhibits cell wall deposition through microtubule disassembly, inducing pits in cell walls. Here, we show that an additional ROP signaling pathway promotes cell wall growth at pit boundaries. Two proteins, Boundary of ROP domain1 (BDR1) and Wallin (WAL), localize to pit boundaries and regulate cell wall growth. WAL interacts with F-actin and promotes actin assembly at pit boundaries while BDR1 is a ROP effector. BDR1 interacts with WAL, suggesting that WAL could be recruited to the plasma membrane by a ROP-dependent mechanism. These results demonstrate that BDR1 and WAL mediate a ROP-actin pathway that shapes pit boundaries. The study reveals a distinct machinery in which two closely associated ROP pathways oppositely regulate cell wall deposition patterns for the establishment of tiny but highly specialized cell wall domains.
Asunto(s)
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Forma de la Célula , Pared Celular/genética , Células Cultivadas , Regulación de la Expresión Génica de las Plantas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microtúbulos/metabolismo , Plantas Modificadas Genéticamente , Xilema/genética , Xilema/metabolismo , Proteínas de Unión al GTP rho/genéticaRESUMEN
Rho-type small GTPases (Rho GTPases) play central roles in various cellular events. Rho GTPases are often activated locally on the plasma membrane, forming plasma membrane domains, which induce downstream signaling. We describe an experimental procedure designed for inducing the production of de novo plasma membrane domains using Arabidopsis ROP11 GTPase. Introduction of ROP11 and its activator and inactivator into the tobacco leaf epidermis leads to formation of ROP11-activated plasma membrane domains on the plasma membrane. Effectors and marker genes can also be introduced alongside ROP11. This reconstruction system allows identifying molecules regulating Rho GTPase polarization.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Microdominios de Membrana/enzimología , Proteínas de Unión al GTP rho/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Microdominios de Membrana/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Membrana Celular/metabolismo , Pared Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Vegetales/metabolismo , Xilema/citología , Proteínas de Unión al GTP rho/metabolismo , Membrana Celular/enzimología , Modelos Teóricos , Células Vegetales/enzimología , Unión ProteicaRESUMEN
Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.
Asunto(s)
Apoptosis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Tallos de la Planta/genética , Sorghum/genética , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/metabolismo , Secuencia de Bases , Carbohidratos/análisis , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Geografía , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Tallos de la Planta/citología , Tallos de la Planta/metabolismo , Homología de Secuencia de Ácido Nucleico , Sorghum/citología , Sorghum/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions.
Asunto(s)
Membrana Celular/fisiología , Microtúbulos/fisiología , Fenómenos Fisiológicos de las PlantasRESUMEN
Proper patterning of the cell wall is essential for plant cell development. Cortical microtubule arrays direct the deposition patterns of cell walls at the plasma membrane. However, the precise mechanism underlying cortical microtubule organization is not well understood. Here, we show that a microtubule-associated protein, CORD1 (CORTICAL MICROTUBULE DISORDERING1), is required for the pitted secondary cell wall pattern of metaxylem vessels in Arabidopsis thaliana Loss of CORD1 and its paralog, CORD2, led to the formation of irregular secondary cell walls with small pits in metaxylem vessels, while overexpressing CORD1 led to the formation of abnormally enlarged secondary cell wall pits. Ectopic expression of CORD1 disturbed the parallel cortical microtubule array by promoting the detachment of microtubules from the plasma membrane. A reconstructive approach revealed that CORD1-induced disorganization of cortical microtubules impairs the boundaries of plasma membrane domains of active ROP11 GTPase, which govern pit formation. Our data suggest that CORD1 promotes cortical microtubule disorganization to regulate secondary cell wall pit formation. The Arabidopsis genome has six CORD1 paralogs that are expressed in various tissues during plant development, suggesting they are important for regulating cortical microtubules during plant development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Tipificación del Cuerpo , Pared Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Xilema/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Secuencia Conservada , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Dominios Proteicos , Xilema/citología , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Diferenciación Celular , Pared Celular/metabolismo , Xilema/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Microtúbulos/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
In vitro xylem differentiation is a powerful technique that can be used to elucidate the process of xylem development that occurs deep inside plant tissues in nature. The experimental procedure described here is designed to induce metaxylem vessel differentiation at exceptionally high frequency and synchronicity using genetically engineered Arabidopsis cell suspensions. By triggering a transcriptional switch, over 80 % of the cells synchronously differentiate into xylem cells within 32 h of treatment with estradiol. Exogenous marker genes can be transiently introduced into the cells by coculturing them with transformed Agrobacterium before inducing xylem differentiation. This system is fast, easy to handle, and highly compatible with molecular and cell biology techniques used to explore xylem cell differentiation.