RESUMEN
Pollen tube growth is an essential step leading to reproductive success in flowering plants, in which vesicular trafficking plays a key role. Vesicular trafficking from endoplasmic reticulum to the Golgi apparatus is mediated by the coat protein complex II (COPII). A key component of COPII is small GTPase Sar1. Five Sar1 isoforms are encoded in the Arabidopsis genome and they show distinct while redundant roles in various cellular and developmental processes, especially in reproduction. Arabidopsis Sar1b is essential for sporophytic control of pollen development while Sar1b and Sar1c are critical for gametophytic control of pollen development. Because functional loss of Sar1b and Sar1c resulted in pollen abortion, whether they influence pollen tube growth was unclear. Here we demonstrate that Sar1b mediates pollen tube growth, in addition to its role in pollen development. Although functional loss of Sar1b does not affect pollen germination, it causes a significant reduction in male transmission and of pollen tube penetration of style. We further show that membrane dynamics at the apex of pollen tubes are compromised by Sar1b loss-of-function. Results presented provide further support of functional complexity of the Sar1 isoforms.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Tubo Polínico/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Regulación de la Expresión Génica de las Plantas , Polen/crecimiento & desarrollo , Polen/genética , Polen/metabolismo , Plantas Modificadas Genéticamente , Germinación/genéticaRESUMEN
Protein S-acylation catalyzed by protein S-acyl transferases (PATs) is a reversible lipid modification regulating protein targeting, stability, and interaction profiles. PATs are encoded by large gene families in plants, and many proteins including receptor-like cytoplasmic kinases (RLCKs) and receptor-like kinases (RLKs) are subject to S-acylation. However, few PATs have been assigned substrates, and few S-acylated proteins have known upstream enzymes. We report that Arabidopsis (Arabidopsis thaliana) class A PATs redundantly mediate pollen tube guidance and participate in the S-acylation of POLLEN RECEPTOR KINASE1 (PRK1) and LOST IN POLLEN TUBE GUIDANCE1 (LIP1), a critical RLK or RLCK for pollen tube guidance, respectively. PAT1, PAT2, PAT3, PAT4, and PAT8, collectively named PENTAPAT for simplicity, are enriched in pollen and show similar subcellular distribution. Functional loss of PENTAPAT reduces seed set due to male gametophytic defects. Specifically, pentapat pollen tubes are compromised in directional growth. We determine that PRK1 and LIP1 interact with PENTAPAT, and their S-acylation is reduced in pentapat pollen. The plasma membrane (PM) association of LIP1 is reduced in pentapat pollen, whereas point mutations reducing PRK1 S-acylation affect its affinity with its interacting proteins. Our results suggest a key role of S-acylation in pollen tube guidance through modulating PM receptor complexes.
RESUMEN
Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes. The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane. Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling. Whereas RopGEFs have been found to be phosphorylated proteins, the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear. We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes. AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes, similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5. Collectively, our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tubo Polínico/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Dominio Catalítico , Citoplasma/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Mutación , Fosforilación , Tubo Polínico/genética , Unión Proteica , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Stomatal movement, critical for photobiosynthesis, respiration, and stress responses, is regulated by many factors, among which abscisic acid (ABA) is critical. Early events of ABA signaling involve Ca2+ influx and an increase of cytoplasmic calcium ([Ca2+]cyt). Positive regulators of this process have been extensively studied, whereas negative regulators are obscure. ABA-induced stomatal closure involves K+ flux and vacuolar convolution. How these processes are connected with Ca2+ is not fully understood. We report that pat10-1, a null mutant of Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is hypersensitive to ABA-induced stomatal closure and vacuolar convolution. A similar phenotype was observed in cbl2;cbl3, the double mutant of CBL2 and CBL3, whose tonoplast association depends on PAT10. Functional loss of the PAT10-CBL2/CBL3 system resulted in enhanced Ca2+ influx and [Ca2+]cyt elevation. Promoting vacuolar K+ accumulation by overexpressing NHX2 suppressed ABA-hypersensitive stomatal closure and vacuolar convolution of the mutants, suggesting that PAT10-CBL2/CBL3 positively mediates vacuolar K+ accumulation. We have identified CBL-interacting protein kinases (CIPKs) that mediate CBL2/CBL3 signaling during ABA-induced stomatal movement. Functional loss of the PAT10-CBL2/3-CIPK9/17 system in guard cells enhanced drought tolerance. We propose that the tonoplast CBL-CIPK complexes form a signaling module that negatively regulates ABA signaling during stomatal movement.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Señalización del Calcio , Estomas de Plantas/fisiología , Ácido Abscísico/farmacología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Sequías , Homeostasis , Estomas de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Potasio/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
Vacuoles are versatile organelles in plant cells, critical for growth and responses to environmental cues. Vacuoles are dynamic tubular structures in pollen tubes, the male gametophytes. Mutations at vacuolar fusion machinery caused male gametophytic lethality by affecting pollen tube growth and guidance, which are critical steps leading to angiosperm reproduction. In comparison, the role of vacuolar trafficking and its cargoes in this process is less understood. In this mini-review, we summarize old and recent findings that indicate the involvement of vacuolar trafficking in pollen tube growth and guidance. We also point at future studies that would provide insights into a key role of vacuolar trafficking and its cargos in pollen tube growth and guidance.
Asunto(s)
Tubo Polínico/metabolismo , Vacuolas/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Tubo Polínico/fisiologíaRESUMEN
Pollen tube growth is an essential step for successful plant reproduction. Vacuolar trafficking and dynamic organization are important for pollen tube growth; however, the key proteins involved in these processes are not well understood. Here, we report that the ADAPTOR PROTEIN-3 (AP-3) complex and its tonoplast cargo PROTEIN S-ACYL TRANSFERASE10 (PAT10) are critical for pollen tube growth in Arabidopsis (Arabidopsis thaliana). AP-3 is a heterotetrameric protein complex consisting of four subunits, δ, ß, µ, and σ. AP-3 regulates tonoplast targeting of several cargoes, such as PAT10. We show that functional loss of any of the four AP-3 subunits reduces plant fertility. In ap-3 mutants, pollen development was normal but pollen tube growth was compromised, leading to reduced male transmission. Functional loss of PAT10 caused a similar reduction in pollen tube growth, suggesting that the tonoplast association of PAT10 mediated by AP-3 is crucial for this process. Indeed, the Ca2+ gradient during pollen tube growth was reduced significantly due to AP-3 loss of function, consistent with the abnormal targeting of CALCINUERIN B-LIKE2 (CBL2) and CBL3, whose tonoplast association depends on PAT10. Furthermore, we show that the pollen tubes of ap-3 mutants have vacuoles with simplified tubules and bulbous structures, indicating that AP-3 affects vacuolar organization. Our results demonstrate a role for AP-3 in plant reproduction and provide insights into the role of vacuoles in polarized cell growth.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Arabidopsis/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Vacuolas/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Señalización del Calcio/genética , Mutación , Plantas Modificadas Genéticamente , Polen/genética , Polen/crecimiento & desarrollo , Tubo Polínico/citología , Tubo Polínico/metabolismo , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.
Asunto(s)
Arabidopsis/genética , Pruebas Genéticas/métodos , Proteínas de Plantas/genética , Tubo Polínico/metabolismo , Vesículas Transportadoras/metabolismo , Vacuolas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Metanosulfonato de Etilo/farmacología , Expresión Génica , Genes Reporteros , Germinación/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis , Imagen Óptica/métodos , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Tubo Polínico/ultraestructura , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semillas/efectos de los fármacos , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Adaptor proteins (APs) mediate protein sorting within endomembrane compartments in eukaryotic cells. AP-3 is an ancient AP complex mediating vacuolar trafficking in different phyla. Only recently, a few tonoplast proteins have been identified as AP-3 cargos in Arabidopsis whereas the function of AP-3 was largely unexplored. Here, we summarize recent advances on AP-3 in Arabidopsis, pointing at the potential roles of AP-3 in plant development and cellular processes.
Asunto(s)
Subunidades del Complejo de Proteínas Adaptadoras/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Modelos BiológicosRESUMEN
Double fertilization in angiosperms requires the delivery of immotile sperm through pollen tubes, which enter embryo sacs to initiate synergid degeneration and to discharge. This fascinating process, called pollen tube reception, involves extensive communications between pollen tubes and synergids, within which few intracellular regulators involved have been revealed. Here, we report that vacuolar acidification in synergids mediated by AP1G and V-ATPases might be critical for pollen tube reception. Functional loss of AP1G or VHA-A, encoding the γ subunit of adaptor protein 1 or the shared component of two endomembrane V-ATPases, respectively, impaired synergid-controlled pollen tube reception and caused partial female sterility. AP1G works in parallel to the plasma membrane-associated receptor FERONIA in synergids, suggesting that synergid-mediated pollen tube reception requires proper sorting of vacuolar cargos by AP1G. Although AP1G did not mediate the targeting of V-ATPases, AP1G loss of function or the expression of AP1G-RNAi compromised vacuolar acidification mediated by V-ATPases, implying their genetic interaction. We propose that vacuolar acidification might represent a distinct cell-death mechanism specifically adopted by the plant phylum, which is critical for synergid degeneration during pollen tube reception.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Arabidopsis/metabolismo , Tubo Polínico/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Arabidopsis/metabolismo , Muerte Celular/fisiología , Membrana Celular/metabolismo , Fertilización/fisiología , Magnoliopsida/metabolismo , Polinización/fisiologíaRESUMEN
Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3δ, whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Complejos Multiproteicos/metabolismo , Vacuolas/metabolismo , Acilación , Membrana Celular/metabolismo , Fluorescencia , Pruebas Genéticas , Ácido Glutámico/metabolismo , Glicina/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Modelos Biológicos , Transporte de Proteínas , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
BACKGROUND: Hybrid vigor is highly valued in the agricultural industry. Male sterility is an important trait for crop breeding. Pollen development is under strict control of both gametophytic and sporophytic factors, and defects in this process can result in male sterility. Both in the dicot Arabidopsis and in the moncot rice, proper timing of programmed cell death (PCD) in the tapetum ensures pollen development. Dynamic ROS levels have been reported to control tapetal PCD, and thus pollen development, in Arabidopsis and rice. However, it was unclear whether it is evolutionarily conserved, as only those two distantly related species were studied. RESULTS: Here, we performed histological analyses of anther development of two economically important dicot species, tobacco and tomato. We identified the same ROS amplitude during anther development in these two species and found that dynamic ROS levels correlate with the initiation and progression of tapetal PCD. We further showed that manipulating ROS levels during anther development severely impaired pollen development, resulting in partial male sterility. Finally, real-time quantitative PCR showed that several tobacco and tomato RBOHs, encoding NADPH oxidases, are preferentially expressed in anthers. CONCLUSION: This study demonstrated evolutionarily conserved ROS amplitude during anther development by examining two commercially important crop species in the Solanaceae. Manipulating ROS amplitude through genetic interference of RBOHs therefore may provide a practical way to generate male sterile plants.
Asunto(s)
Nicotiana/citología , Células Vegetales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Solanum lycopersicum/citología , Muerte Celular , Flores/crecimiento & desarrollo , Flores/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/genética , Polen/crecimiento & desarrollo , Polen/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Vacuolar trafficking routes and their regulators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER-to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V-ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vacuolas/metabolismo , Meristema/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Polar growth of root hairs is critical for plant survival and requires fine-tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S-acyl transferases (PATs), mediating the S-acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site-directed mutagenesis and genetic analysis with related root-hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S-acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild-type but not an enzyme-inactive PAT4. Membrane-associated ROP2 was significantly reduced in pat4, similar to S-acylation-deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4-mediated S-acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Actinas/metabolismo , Aciltransferasas/genética , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Transducción de SeñalRESUMEN
Prenylation, the post-translational attachment of prenyl groups to substrate proteins, can affect their distribution and interactomes. Arabidopsis PLURIPETALA (PLP) encodes the shared α subunit of two heterodimeric protein isoprenyltransferases, whose functional loss provides a unique opportunity to study developmental and cellular processes mediated by its prenylated substrates, such as ROP GTPases. As molecular switches, the distribution and activation of ROPs are mediated by various factors, including guanine nucleotide exchange factors, GTPase activating proteins, guanine nucleotide dissociation inhibitors (RhoGDIs), prenylation, and S-acylation. However, how these factors together ensure that dynamic ROP signalling is still obscure. We report here that a loss-of-function allele of PLP resulted in cytoplasmic accumulation of ROP2 in root hairs and reduced its stability. Consequently, two downstream events of ROP signalling, i.e. actin microfilament (MF) organization and the production of reactive oxygen species (ROS), were compromised. Genetic, cytological and biochemical evidence supports an additive interaction between prenylation and RhoGDI1/SCN1 in ROP2 distribution and stability whereas PLP acts synergistically with the protein S-acyl transferase TIP GROWTH DEFECTIVE1 during root hair growth. By using root hair growth as a model system, we uncovered complex interactions among prenylation, RhoGDIs, and S-acylation in dynamic ROP signalling.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Unión al GTP/metabolismo , Acilación , Aciltransferasas/genética , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Catión/genética , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Transducción de Señal , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Homeostasis , Mutación , Polen/genética , Polen/crecimiento & desarrollo , Polen/fisiología , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/fisiología , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/genéticaRESUMEN
BACKGROUND: Protein palmitoylation, which is critical for membrane association and subcellular targeting of many signaling proteins, is catalyzed mainly by protein S-acyl transferases (PATs). Only a few plant proteins have been experimentally verified to be subject to palmitoylation, such as ROP GTPases, calcineurin B like proteins (CBLs), and subunits of heterotrimeric G proteins. However, emerging evidence from palmitoyl proteomics hinted that protein palmitoylation as a post-translational modification might be widespread. Nonetheless, due to the large number of genes encoding PATs and the lack of consensus motifs for palmitoylation, progress on the roles of protein palmitoylation in plants has been slow. RESULTS: We combined pharmacological and genetic approaches to examine the role of protein palmitoylation in root hair growth. Multiple PATs from different endomembrane compartments may participate in root hair growth, among which the Golgi-localized PAT24/TIP GROWTH DEFECTIVE1 (TIP1) plays a major role while the tonoplast-localized PAT10 plays a secondary role in root hair growth. A specific inhibitor for protein palmitoylation, 2-bromopalmitate (2-BP), compromised root hair elongation and polarity. Using various probes specific for cellular processes, we demonstrated that 2-BP impaired the dynamic polymerization of actin microfilaments (MF), the asymmetric plasma membrane (PM) localization of phosphatidylinositol (4,5)-bisphosphate (PIP2), the dynamic distribution of RabA4b-positive post-Golgi secretion, and endocytic trafficking in root hairs. CONCLUSIONS: By combining pharmacological and genetic approaches and using root hairs as a model, we show that protein palmitoylation, regulated by protein S-acyl transferases at different endomembrane compartments such as the Golgi and the vacuole, is critical for the polar growth of root hairs in Arabidopsis. Inhibition of protein palmitoylation by 2-BP disturbed key intracellular activities in root hairs. Although some of these effects are likely indirect, the cytological data reported here will contribute to a deep understanding of protein palmitoylation during tip growth in plants.
Asunto(s)
Aciltransferasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Lipoilación , Palmitatos/metabolismo , Aciltransferasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismoAsunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Membrana Celular/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Tubo Polínico/citología , Tubo Polínico/crecimiento & desarrollo , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins.