RESUMO
Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.
RESUMO
Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5 (ABI5) is a central regulator of ABA signaling. ABI5 BINDING PROTEIN 1 (AFP1) interacts with ABI5 and facilitates its 26S-proteasome-mediated degradation, although the detailed mechanism has remained unclear. Here, we report that an ABA-responsive U-box E3 ubiquitin ligase, PLANT U-BOX 35 (PUB35), physically interacts with AFP1 and ABI5. PUB35 directly ubiquitinated ABI5 in a bacterially reconstituted ubiquitination system and promoted ABI5 protein degradation in vivo. ABI5 degradation was enhanced by AFP1 in response to ABA treatment. Phosphorylation of the T201 and T206 residues in ABI5 disrupted the ABI5-AFP1 interaction and affected the ABI5-PUB35 interaction and PUB35-mediated degradation of ABI5 in vivo. Genetic analysis of seed germination and seedling growth showed that pub35 mutants were hypersensitive to ABA as well as to salinity and osmotic stresses, whereas PUB35 overexpression lines were hyposensitive. Moreover, abi5 was epistatic to pub35, whereas the pub35-2 afp1-1 double mutant showed a similar ABA response to the two single mutants. Together, our results reveal a PUB35-AFP1 module involved in fine-tuning ABA signaling through ubiquitination and 26S-proteasome-mediated degradation of ABI5 during seed germination and seedling growth.
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The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.
Assuntos
Arabidopsis , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Endossomos/metabolismo , Vacúolos/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Arabidopsis/metabolismo , Nexinas de Classificação/metabolismoRESUMO
Lipid droplets (LDs) stored during seed development are mobilized and provide essential energy and lipids to support seedling growth upon germination. Triacylglycerols (TAGs) are the main neutral lipids stored in LDs. The lipase SUGAR DEPENDENT 1 (SDP1), which hydrolyzes TAGs in Arabidopsis thaliana, is localized on peroxisomes and traffics to the LD surface through peroxisomal extension, but the underlying mechanism remains elusive. Here, we report a previously unknown function of a plant-unique endosomal sorting complex required for transport (ESCRT) component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) in regulating peroxisome/SDP1-mediated LD turnover in Arabidopsis. We showed that LD degradation was impaired in germinating free1 mutant; moreover, the tubulation of SDP1- or PEROXIN 11e (PEX11e)-marked peroxisomes and the migration of SDP1-positive peroxisomes to the LD surface were altered in the free1 mutant. Electron tomography analysis showed that peroxisomes failed to form tubules to engulf LDs in free1, unlike in the wild-type. FREE1 interacted directly with both PEX11e and SDP1, suggesting that these interactions may regulate peroxisomal extension and trafficking of the lipase SDP1 to LDs. Taken together, our results demonstrate a pivotal role for FREE1 in LD degradation in germinating seedlings via regulating peroxisomal tubulation and SDP1 targeting.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Plântula/metabolismo , Peroxissomos/metabolismo , Proteínas de Arabidopsis/metabolismo , Gotículas Lipídicas/metabolismo , Lipase/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lipídeos , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Plantas/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismoRESUMO
The transition from vegetative to reproductive growth, known as flowering, is a critical developmental process in flowering plants to ensure reproductive success. This process is strictly controlled by various internal and external cues; however, the underlying molecular regulatory mechanisms need to be further characterized. Here, we report a plant-specific protein, FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13), which functions as a hitherto unknown negative modulator of flowering time in Arabidopsis thaliana. Biochemical analysis showed that FLZ13 directly interacts with FLOWERING LOCUS C (FLC), a major flowering repressor, and that FLZ13 largely depends on FLC to repress the transcription of two core flowering integrators: FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. In addition, FLZ13 works together with ABSCISIC ACID INSENSITIVE 5 to activate FLC expression to delay flowering. Taken together, our findings suggest that FLZ13 is an important component of the gene regulatory network for flowering time control in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flores , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismoRESUMO
As a fundamental metabolic pathway, autophagy plays important roles in plant growth and development, particularly under stress conditions. A set of autophagy-related (ATG) proteins is recruited for the formation of a double-membrane autophagosome. Among them, the essential roles of ATG2, ATG18, and ATG9 have been well established in plant autophagy via genetic analysis; however, the underlying molecular mechanism for ATG2 in plant autophagosome formation remains poorly understood. In this study, we focused on the specific role of ATG2 in the trafficking of ATG18a and ATG9 during autophagy in Arabidopsis (Arabidopsis thaliana). Under normal conditions, YFP-ATG18a proteins are partially localized on late endosomes and translocated to ATG8e-labeled autophagosomes upon autophagic induction. Real-time imaging analysis revealed sequential recruitment of ATG18a on the phagophore membrane, showing that ATG18a specifically decorated the closing edges and finally disassociated from the completed autophagosome. However, in the absence of ATG2, most of the YFP-ATG18a proteins are arrested on autophagosomal membranes. Ultrastructural and 3D tomography analysis showed that unclosed autophagosome structures are accumulated in the atg2 mutant, displaying direct connections with the endoplasmic reticulum membrane and vesicular structures. Dynamic analysis of ATG9 vesicles suggested that ATG2 depletion also affects the association between ATG9 vesicles and the autophagosomal membrane. Furthermore, using interaction and recruitment analysis, we mapped the interaction relationship between ATG2 and ATG18a, implying a possible role of ATG18a in recruiting ATG2 and ATG9 to the membrane. Our findings unveil a specific role of ATG2 in coordinating ATG18a and ATG9 trafficking to mediate autophagosome closure in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Saccharomyces cerevisiae , Autofagossomos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Autofagia/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Autophagy, a highly conserved quality control mechanism, is essential for maintaining cellular homeostasis and healthy growth of plants. Compared with extensive research in the cytoplasmic control of autophagy, studies regarding the nuclear events involved in the regulation of plant autophagy are just beginning to emerge. Accumulating evidence reveals a coordinated expression of plant autophagy genes in response to diverse developmental states and growth conditions. Here, we summarize recent progress in the identification of tightly controlled transcription factors and histone marks associated with the autophagic process in plants, and propose several modules, consisting of transcription regulators and epigenetic modifiers, as important nuclear players that could contribute to both short-term and long-term controls of plant autophagy at the transcriptional and post-transcriptional levels.
Assuntos
Autofagia , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Plantas/genética , Transcrição Gênica , Transdução de SinaisRESUMO
Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagossomos/metabolismo , Autofagia , Plantas/metabolismo , Proteínas de Transporte/metabolismo , Arabidopsis/metabolismo , Mamíferos , Proteínas de Arabidopsis/metabolismoRESUMO
The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.
Assuntos
Endossomos , Plantas , Plantas/metabolismo , Endossomos/metabolismo , Vacúolos/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Proteico , Complexo de Golgi/metabolismo , Rede trans-Golgi/metabolismoRESUMO
The FYVE domain protein required for endosomal sorting 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport machinery, plays an essential role in endosomal trafficking. Moreover, FREE1 also functions as an important negative regulator in abscisic acid (ABA) signalling. Multiple phosphorylations and ubiquitination sites have been identified in FREE1, hence unveiling the factors involved in posttranslational regulation of FREE1 is critical for comprehensively understanding FREE1-related regulatory networks during plant growth. Here, we demonstrate that plant-specific casein kinase I members MUT9-like kinases 1-4 (MLKs 1-4)/Arabidopsis EL1-like 1-4 interact with and phosphorylate FREE1 at serine residue S582, thereby modulating the nuclear accumulation of FREE1. Consequently, mutation of S582 to non-phosphorylable residue results in reduced nuclear localization of FREE1 and enhanced ABA response. In addition, mlk123 and mlk134 triple mutants accumulate less FREE1 in the nucleus and display hypersensitive responses to ABA treatment, whereas overexpression of the nuclear-localized FREE1 can restore the ABA sensitivity of seedling establishment in mlks triple mutants. Collectively, our study demonstrates a previously unidentified function of MLKs in attenuating ABA signalling in the nucleus by regulating the phosphorylation and nuclear accumulation of FREE1.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Plântula/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Autophagy is a highly conserved, self-digestion process that is essential for plant adaptations to various environmental stresses. Although the core components of autophagy in plants have been well established, the molecular basis for its transcriptional regulation remains to be fully characterized. In this study, we demonstrate that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS-box family transcription factor that determines flowering transition in Arabidopsis, functions as a transcriptional repressor of autophagy. EMSAs, ChIP-qPCR assays, and dual-luciferase receptor assays showed that SOC1 can bind to the promoters of ATG4b, ATG7, and ATG18c via the conserved CArG box. qRT-PCR analysis showed that the three ATG genes ATG4b, ATG7, and ATG18c were up-regulated in the soc1-2 mutant. In line with this, the mutant also displayed enhanced autophagy activity, as revealed by increased autophagosome formation and elevated autophagic flux compared with the wild type. More importantly, SOC1 negatively affected the tolerance of plants to long-term carbon starvation, and this process requires a functional autophagy pathway. Finally, we found that SOC1 was repressed upon carbon starvation at both the transcriptional and protein levels. Overall, our study not only uncovers an important transcriptional mechanism that contributes to the regulation of plant autophagy in response to nutrient starvation, but also highlights novel cellular functions of the flowering integrator SOC1.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Carbono/metabolismo , Flores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Autofagia/genéticaRESUMO
ENDOMEMBRANE PROTEIN 70 (EMP70) proteins constitute a 12-member superfamily in Arabidopsis thaliana, and are the most abundant protein species in plant Golgi proteomes. However, the physiological functions of EMPs in plants remain largely unknown. Here we have demonstrated that two AtEMP12 T-DNA insertion mutants are sensitive to ER (endoplasmic reticulum) stress as induced by tunicamycin and dithiothreitol treatments. Interestingly, the unfolded protein response (UPR) is constitutively activated in the knockout mutant emp12-1 under normal growth conditions, suggesting that the activation is a result of insufficient chaperones in the ER to aid protein folding. Indeed, we have further shown that BiP is secreted into the apoplast in emp12-1, while the K/HDEL receptor ERD2a, which regulates BiP trafficking, is exclusively localized in the ER in emp12-1, instead of its known ER-Golgi dual-localization. Given an enhanced retrograde transport of ERD2a, along with less dimerized receptor formed in the absence of EMP12, ERD2a may be prematurely returned to the ER without its bound ligands. Therefore, we propose that EMP12 may act as a novel regulator of the K/HDEL receptor to ensure an effective retrograde transport of K/HDEL ligands.
RESUMO
FYVE domain protein required for endosomal sorting1 (FREE1), a plant-specific endosomal sorting complex required for transport-I component, is essential for the biogenesis of multivesicular bodies (MVBs), vacuolar degradation of membrane protein, cargo vacuolar sorting, autophagic degradation, and vacuole biogenesis in Arabidopsis (Arabidopsis thaliana). Here, we report the characterization of RESURRECTION1 (RST1) as a suppressor of free1 that, when mutated as a null mutant, restores the normal MVB and vacuole formation of a FREE1-RNAi knockdown line and consequently allows survival. RST1 encodes an evolutionarily conserved multicellular organism-specific protein, which contains two Domain of Unknown Function 3730 domains, showing no similarity to known proteins, and predominantly localizes in the cytosol. The depletion of FREE1 causes substantial accumulation of RST1, and transgenic Arabidopsis plants overexpressing RST1 display retarded seedling growth with dilated MVBs, and inhibition of endocytosed FM4-64 dye to the tonoplast, suggesting that RST1 has a negative role in vacuolar transport. Consistently, enhanced endocytic degradation of membrane vacuolar cargoes occurs in the rst1 mutant. Further transcriptomic comparison of rst1 with free1 revealed a negative association between gene expression profiles, demonstrating that FREE1 and RST1 have antagonistic functions. Thus, RST1 is a negative regulator controlling membrane protein homeostasis and FREE1-mediated functions in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Corpos Multivesiculares/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico/genética , Interferência de RNA , Plântula/crescimento & desenvolvimento , Transcriptoma , Proteínas de Transporte Vesicular/genéticaRESUMO
Under nutrient and energy-limiting conditions, plants up-regulate sophisticated catabolic pathways such as autophagy to remobilize nutrients and restore energy homeostasis. Autophagic flux is tightly regulated under these circumstances through the AuTophaGy-related1 (ATG1) kinase complex, which relays upstream nutrient and energy signals to the downstream components that drive autophagy. Here, we investigated the role(s) of the Arabidopsis (Arabidopsis thaliana) ATG1 kinase during autophagy through an analysis of a quadruple mutant deficient in all four ATG1 isoforms. These isoforms appear to act redundantly, including the plant-specific, truncated ATG1t variant, and like other well-characterized atg mutants, homozygous atg1abct quadruple mutants display early leaf senescence and hypersensitivity to nitrogen and fixed-carbon starvations. Although ATG1 kinase is essential for up-regulating autophagy under nitrogen deprivation and short-term carbon starvation, it did not stimulate autophagy under prolonged carbon starvation. Instead, an ATG1-independent response arose requiring phosphatidylinositol-3-phosphate kinase (PI3K) and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), possibly through phosphorylation of the ATG6 subunit within the PI3K complex by the catalytic KIN10 subunit of SnRK1. Together, our data connect ATG1 kinase to autophagy and reveal that plants engage multiple pathways to activate autophagy during nutrient stress, which include the ATG1 route as well as an alternative route requiring SnRK1 and ATG6 signaling.plantcell;31/12/2973/FX1F1fx1.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Carbono/deficiência , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Compostos de Amônio/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/química , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carbono/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Nitrogênio/deficiência , Nitrogênio/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Isoformas de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide-binding capabilities. Nonetheless, the function of most plant phosphoinositide-binding proteins in endosomal trafficking remains elusive. Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide-binding proteins and first identified fyve4-1 as a mutant with a hypersensitive response to high-boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1-GFP. FYVE4 encodes a plant-unique, FYVE domain-containing protein that interacts with SNF7, a core component of ESCRT-III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late-acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4-1, snf7b and lip5 single mutants were viable, the fyve4-1 snf7b and fyve4-1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins. Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis , Complexos Endossomais de Distribuição Requeridos para Transporte , Arabidopsis/genética , Arabidopsis/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Desenvolvimento Vegetal , Transporte Proteico , Vacúolos/metabolismoRESUMO
Multivesicular body (MVB)-mediated endosomal sorting and macroautophagy are the main pathways mediating the transport of cellular components to the vacuole and are essential for maintaining cellular homeostasis. The interplay of these two pathways remains poorly understood in plants. In this study, we show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT), essential for MVB biogenesis and plant growth, can be transported to the vacuole for degradation in response to iron deficiency. The vacuolar transport of ubiquitinated FREE1 protein is mediated by the autophagy pathway. As a consequence, the autophagy deficient mutants, atg5-1 and atg7-2, accumulate more endogenous FREE1 protein and display hypersensitivity to iron deficiency. Furthermore, under iron-deficient growth condition autophagy related genes are upregulated to promote the autophagic degradation of FREE1, thereby possibly relieving the repressive effect of FREE1 on iron absorption. Collectively, our findings demonstrate a unique regulatory mode of protein turnover of the ESCRT machinery through the autophagy pathway to respond to iron deficiency in plants.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/genética , Ferro/metabolismo , Proteínas de Transporte Vesicular/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Autofagia , Transporte Biológico , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Mutação , Proteólise , UbiquitinaçãoRESUMO
FCS-like zinc finger family proteins (FLZs), a class of plant-specific scaffold of SnRK1 complex, are involved in the regulation of various aspects of plant growth and stress responses. Most information of FLZ family genes was obtained from the studies in Arabidopsis thaliana, whereas little is known about the potential functions of FLZs in crop plants. In this study, 37 maize FLZ (ZmFLZ) genes were identified to be asymmetrically distributed on 10 chromosomes and can be divided into three subfamilies. Protein interaction and subcellular localization assays demonstrated that eight typical ZmFLZs interacted and partially co-localized with ZmKIN10, the catalytic α-subunit of the SnRK1 complex in maize leaf mesophyll cells. Expression profile analysis revealed that several ZmFLZs were differentially expressed across various tissues and actively responded to diverse abiotic stresses. In addition, ectopic overexpression of ZmFLZ25 in Arabidopsis conferred hypersensitivity to exogenous abscisic acid (ABA) and triggered higher expression of ABA-induced genes, pointing to the positive regulatory role of ZmFLZ25 in plant ABA signaling, a scenario further evidenced by the interactions between ZmFLZ25 and ABA receptors. In summary, these data provide the most comprehensive information on FLZ family genes in maize, and shed light on the biological function of ZmFLZ25 in plant ABA signaling.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Plantas/genética , Zea mays/genética , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Estudo de Associação Genômica Ampla , Família Multigênica , Especificidade de Órgãos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Fisiológico/genética , Zea mays/efeitos dos fármacos , Dedos de Zinco/genéticaRESUMO
Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.