RESUMEN
Extreme environmental conditions eventually limit plant growth [J. R. Dinneny, Annu. Rev. Cell Dev. Biol. 35, 1-19 (2019), N. Gigli-Bisceglia, C. Testerink, Curr. Opin. Plant Biol. 64, 102120 (2021)]. Here, we reveal a mechanism that enables multiple external cues to get integrated into auxin-dependent growth programs in Arabidopsis thaliana. Our forward genetics approach on dark-grown hypocotyls uncovered that an imbalance in membrane lipids enhances the protein abundance of PIN-LIKES (PILS) [E. Barbez et al., Nature 485, 119 (2012)] auxin transport facilitators at the endoplasmic reticulum (ER), which thereby limits nuclear auxin signaling and growth rates. We show that this subcellular response relates to ER stress signaling, which directly impacts PILS protein turnover in a tissue-dependent manner. This mechanism allows PILS proteins to integrate environmental input with phytohormone auxin signaling, contributing to stress-induced growth adaptation in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Plantas/metabolismo , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica de las PlantasRESUMEN
Multiple internal and external signals modulate the metabolism, intercellular transport and signaling of the phytohormone auxin. Considering this complexity, it remains largely unknown how plant cells monitor and ensure the homeostasis of auxin responses. PIN-LIKES (PILS) intracellular auxin transport facilitators at the endoplasmic reticulum are suitable candidates to buffer cellular auxin responses because they limit nuclear abundance and signaling of auxin. We used forward genetics to identify gloomy and shiny pils (gasp) mutants that define the PILS6 protein abundance in a post-translational manner. Here, we show that GASP1 encodes an uncharacterized RING/U-box superfamily protein that impacts on auxin signaling output. The low auxin signaling in gasp1 mutants correlates with reduced abundance of PILS5 and PILS6 proteins. Mechanistically, we show that high and low auxin conditions increase and reduce PILS6 protein levels, respectively. Accordingly, non-optimum auxin concentrations are buffered by alterations in PILS6 abundance, consequently leading to homeostatic auxin output regulation. We envision that this feedback mechanism provides robustness to auxin-dependent plant development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/fisiología , Retroalimentación , Regulación de la Expresión Génica de las Plantas , Homeostasis , Ácidos Indolacéticos/metabolismoRESUMEN
The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.
Asunto(s)
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Endosomas , Estomas de Plantas , Transducción de Señal , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endosomas/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Estomas de Plantas/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Sequías , Mutación/genética , Proteolisis , Transporte de ProteínasRESUMEN
Cellular elongation requires the defined coordination of intra- and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsis thaliana We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read-out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine-rich repeat extensins (LRXs) with the receptor-like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane-localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Fosfotransferasas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Vacuolas/metabolismo , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas Repetidas Ricas en Leucina , Desarrollo de la PlantaRESUMEN
Temperature modulates growth and development throughout the entire lifecycle of a plant. High temperature (HT) triggers the auxin biosynthesis-dependent growth in aerial tissues. On the other hand, the contribution of auxin to HT-induced root growth is currently under debate. Here we show that the putative intracellular auxin carrier PIN-LIKES 6 (PILS6) is a negative regulator of organ growth and that its abundance is highly sensitive to HT. PILS6 localizes to the endoplasmic reticulum and limits the nuclear availability of auxin, consequently reducing the auxin signaling output. HT represses the PILS6 protein abundance, which impacts on PILS6-dependent auxin signaling in roots and root expansion. Accordingly, we hypothesize that PILS6 is part of an alternative mechanism linking HT to auxin responses in roots.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Raíces de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Calor , Proteínas de Transporte de Membrana/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Transducción de Señal , TemperaturaRESUMEN
Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Represoras/metabolismo , Plantones/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Represoras/genética , Plantones/genética , Plantones/metabolismo , Transducción de SeñalRESUMEN
Phosphatidylinositol (PtdIns) is a structural phospholipid that can be phosphorylated into various lipid signaling molecules, designated polyphosphoinositides (PPIs). The reversible phosphorylation of PPIs on the 3, 4, or 5 position of inositol is performed by a set of organelle-specific kinases and phosphatases, and the characteristic head groups make these molecules ideal for regulating biological processes in time and space. In yeast and mammals, PtdIns3P and PtdIns(3,5)P2 play crucial roles in trafficking toward the lytic compartments, whereas the role in plants is not yet fully understood. Here we identified the role of a land plant-specific subgroup of PPI phosphatases, the suppressor of actin 2 (SAC2) to SAC5, during vacuolar trafficking and morphogenesis in Arabidopsis thaliana. SAC2-SAC5 localize to the tonoplast along with PtdIns3P, the presumable product of their activity. In SAC gain- and loss-of-function mutants, the levels of PtdIns monophosphates and bisphosphates were changed, with opposite effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles was defective. Moreover, multiple sac knockout mutants had an increased number of smaller storage and lytic vacuoles, whereas extralarge vacuoles were observed in the overexpression lines, correlating with various growth and developmental defects. The fragmented vacuolar phenotype of sac mutants could be mimicked by treating wild-type seedlings with PtdIns(3,5)P2, corroborating that this PPI is important for vacuole morphology. Taken together, these results provide evidence that PPIs, together with their metabolic enzymes SAC2-SAC5, are crucial for vacuolar trafficking and for vacuolar morphology and function in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Cartilla de ADN/genética , Evolución Molecular , Microscopía Electrónica de Transmisión , Fenotipo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas Fosfatasas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacuolas/metabolismo , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Polaridad Celular/genética , Dineínas Citoplasmáticas/genética , Proteínas Munc18/genética , Oxidorreductasas de Alcohol/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Membrana Celular/genética , Membrana Celular/metabolismo , Endosomas/genética , Endosomas/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Proteínas Munc18/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Transporte de Proteínas/genética , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismoRESUMEN
Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging-based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Red trans-Golgi/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Brefeldino A/farmacología , Membrana Celular/ultraestructura , Endosomas/ultraestructura , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Fluorescentes Verdes , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación Missense , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantas Modificadas Genéticamente , Transporte de Proteínas , Plantones/efectos de los fármacos , Plantones/genética , Plantones/metabolismo , Plantones/ultraestructura , Red trans-Golgi/ultraestructuraRESUMEN
Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1-green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brefeldino A/farmacología , Endocitosis/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Proteínas de Transporte de Membrana/metabolismo , Desarrollo de la Planta/efectos de los fármacos , Factores de Ribosilacion-ADP/metabolismo , Alelos , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Clonación Molecular , Epistasis Genética/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Proteínas de Transporte de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Mutación/genética , Transporte de Proteínas/efectos de los fármacosRESUMEN
Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker-based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative ß-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 ß adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 ß in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Vacuolas/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clonación Molecular , Mutación , Transporte de ProteínasRESUMEN
A Correction to this paper has been published: https://doi.org/10.1038/s41477-021-00924-y.
RESUMEN
Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that BR signaling controls PIN-LIKES (PILS)-dependent nuclear abundance of auxin. We performed a forward genetic screen for imperial pils (imp) mutants that enhance the overexpression phenotypes of PILS5 putative intracellular auxin transport facilitator. Here, we report that the imp1 mutant is defective in the BR-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Our set of data reveals that BR signaling transcriptionally and post-translationally represses the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Brasinoesteroides/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Transporte de Membrana/genética , Neoplasias Basocelulares , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/fisiología , Proteínas Quinasas/genéticaRESUMEN
The phytohormone auxin induces or represses growth depending on its concentration and the underlying tissue type. However, it remains unknown how auxin signalling is modulated to allow tissues transiting between repression and promotion of growth. Here, we used apical hook development as a model for growth transitions in plants. A PIN-FORMED (PIN)-dependent intercellular auxin transport module defines an auxin maximum that is causal for growth repression during the formation of the apical hook. Our data illustrate that growth transition for apical hook opening is largely independent of this PIN module, but requires the PIN-LIKES (PILS) putative auxin carriers at the endoplasmic reticulum. PILS proteins reduce nuclear auxin signalling in the apical hook, leading to the de-repression of growth and the onset of hook opening. We also show that the phytochrome (phy) B-reliant light-signalling pathway directly regulates PILS gene activity, thereby enabling light perception to repress nuclear auxin signalling and to control growth. We propose a novel mechanism, in which PILS proteins allow external signals to alter tissue sensitivity to auxin, defining differential growth rates.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Ácidos Indolacéticos/metabolismo , Luz , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal/efectos de la radiación , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Fitocromo B/metabolismoRESUMEN
Plant growth relates to gravity, ensuring that roots grow downwards into the soil and shoots expand aerially. The phytohormone auxin mediates tropistic growth responses, such as root gravitropism. Gravity perception in the very tip of the roots triggers carrier-dependent, asymmetric redistribution of auxin, leading to differential auxin responses and growth regulation at the upper and lower root flanks. This cellular, asymmetry-breaking event will eventually lead to root bending towards the gravity vector. Here, we show how to investigate auxin signaling and auxin carrier dynamics during root gravitropic response, using a chambered cover glass in combination with a confocal live cell imaging approach. To exemplify this method, we used established lines expressing transcriptional and translational green fluorescent protein (GFP) fusions to the auxin responsive promoter element DR5rev and the prominent auxin carrier PIN-FORMED2 (PIN2), respectively. Transgenic seedlings were placed and grown in the chambered cover glasses, enabling defined gravitropic stimulations prior to imaging. This method is optimal for inverted microscopes and significantly reduces stressful manipulations during specimen preparation.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Arabidopsis/crecimiento & desarrollo , Gravitación , Gravitropismo , Proteínas Fluorescentes Verdes , Raíces de Plantas/metabolismo , Transducción de SeñalRESUMEN
Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Compartimento Celular , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endocitosis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Mutación , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS) putative auxin carriers localize to the endoplasmic reticulum (ER) and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and, subsequently, affect nuclear auxin signaling. Here, we investigate sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions.
RESUMEN
Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative δ subunit of the AP-3 complex. pat4 and pat2, a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 ß, as well as dominant negative AP-3 µ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development, but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 ß and AP-3 δ subunits. Furthermore, both proteins are closely linked with putative AP-3 µ and σ subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells.