RESUMEN
It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells (PCs). However, the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood. Here, we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9 (ADF9) is required for the morphogenesis of PC, which is negatively regulated by the R2R3 MYELOBLASTOSIS (MYB) transcription factor MYB52. In adf9 mutants, the lobe number of cotyledon PCs was significantly reduced, while the average lobe length did not differ significantly compared to that of wild type (Col-0), except for the variations in cell area and circularity, whereas the PC shapes in ADF9 overexpression seedlings showed different results. ADF9 decorated actin filaments, and colocalized with plasma membrane. The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased. In addition, MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression. The myb52-2 mutant showed increased lobe number and cell area, reduced cell circularity of PCs, and the PC phenotypes were suppressed when ADF9 was knocked out. Taken together, our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.
RESUMEN
Plants must cope with diverse environmental stresses during growth and development, among which drought is one of the most concerning global threats. Recent studies have shown that the disassembly of the microtubule cytoskeleton plays an essential role during ABA-induced stomatal closure in response to drought stress. To facilitate studies on the mechanisms of ABA-induced microtubule rearrangement during stomatal closure, we describe procedures for observing guard cells treated with ABA, visualizing the microtubule cytoskeleton in guard cells, and their subsequent quantitative analysis. We include both representative images and the quantification results to illustrate these experiments.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Ácido Abscísico/farmacología , Estomas de Plantas , Microtúbulos , MutaciónRESUMEN
Hypocotyl elongation is an important morphological response during plant thermomorphogenesis. Multiple studies indicate that the transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a key regulator of high temperature-induced hypocotyl elongation. However, the underlying cellular mechanisms regarding PIF4-mediated hypocotyl elongation are largely unclear. In this study, we found that PIF4 regulates the PLANT U-BOX TYPE E3 UBIQUITIN LIGASE 31 (PUB31)-SPIRAL1 (SPR1) module and alters cortical microtubule reorganization to promote hypocotyl cell elongation during Arabidopsis thaliana (Arabidopsis) thermomorphogenesis. SPR1 loss-of-function mutants exhibit much shorter hypocotyls when grown at 28 °C, indicating a positive role for SPR1 in high ambient temperature-induced hypocotyl elongation. High ambient temperature induces SPR1 expression in a PIF4-dependent manner, and stabilizes SPR1 protein to mediate microtubule reorganization. Further investigation showed that PUB31 interacts with and ubiquitinates SPR1. In particular, the ubiquitinated effect on SPR1 was moderately decreased at high temperature, which was due to the direct binding of PIF4 to the PUB31 promoter and down-regulating its expression. Thus, this study reveals a mechanism in which PIF4 induces SPR1 expression and suppresses PUB31 expression, resulting in the accumulation and stabilization of SPR1 protein, and further promoting hypocotyl cell elongation by altering cortical microtubule organization during Arabidopsis thermomorphogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hipocótilo/metabolismo , Fitocromo/metabolismo , Temperatura , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Tubulin folding cofactors (TFCs) are required for tubulin folding, α/ß tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Tubulina (Proteína) , Animales , Tubulina (Proteína)/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Zea mays/genética , Zea mays/metabolismo , Microtúbulos/metabolismo , División Celular , Homeostasis , MamíferosRESUMEN
Tight control of lateral root (LR) initiation is vital for root system architecture and function. Regulation of cortical microtubule reorganization is involved in the asymmetric radial expansion of founder cells during LR initiation in Arabidopsis (Arabidopsis thaliana). However, critical genetic evidence on the role of microtubules in LR initiation is lacking and the mechanisms underlying this regulation are poorly understood. Here, we found that the previously uncharacterized microtubule-stabilizing protein TPX2-LIKE5 (TPXL5) participates in LR initiation, which is finely regulated by the transcription factor ELONGATED HYPOCOTYL5 (HY5). In tpxl5 mutants, LR density was decreased and more LR primordia (LRPs) remained in stage I, indicating delayed LR initiation. In particular, the cell width in the peripheral domain of LR founder cells after the first asymmetric cell division was larger in tpxl5 mutants than in the wild-type. Consistently, ordered transverse cortical microtubule arrays were not well generated in tpxl5 mutants. In addition, HY5 directly targeted the promoter of TPXL5 and downregulated TPXL5 expression. The hy5 mutant exhibited higher LR density and fewer stage I LRPs, indicating accelerated LR initiation. Such phenotypes were partially suppressed by TPXL5 knockout. Taken together, our data provide genetic evidence supporting the notion that cortical microtubules are essential for LR initiation and unravel a molecular mechanism underlying HY5 regulation of TPXL5-mediated microtubule reorganization and cell remodeling during LR initiation.
Asunto(s)
Proteínas de Arabidopsis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas del Citoesqueleto , Raíces de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , División Celular Asimétrica , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica de las Plantas , Microtúbulos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Microtúbulos , Proteínas Quinasas , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estomas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Agua/metabolismoRESUMEN
Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca2+ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca2+ signaling was blocked by BAPTA-AM or LaCl3 , microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Citosol/metabolismo , Microtúbulos/metabolismo , Estrés Salino , Plantones/metabolismoRESUMEN
In multicellular and even single-celled organisms, individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation. Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes. Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project. In plant science, network analysis has similarly been applied to study the connectivity of plant components at the molecular, subcellular, cellular, organic, and organism levels. Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype. In this review, we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities. We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants. Finally, we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.
Asunto(s)
Diagnóstico por Imagen , Modelos Biológicos , Células Vegetales/fisiología , Fenómenos Fisiológicos de las Plantas , Biología de Sistemas , Genotipo , FenotipoRESUMEN
Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Microtúbulos/metabolismo , Estomas de Plantas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Microtúbulos/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Estomas de Plantas/citología , Estomas de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The unique apical hook in dicotyledonous plants protects the shoot apical meristem and cotyledons when seedlings emerge through the soil. Its formation involves differential cell growth under the coordinated control of plant hormones, especially ethylene and auxin. Microtubules are essential players in plant cell growth that are regulated by multiple microtubule-associated proteins (MAPs). However, the role and underlying mechanisms of MAP-microtubule modules in differential cell growth are poorly understood. In this study, we found that the previously uncharacterized Arabidopsis MAP WAVE-DAMPENED2-LIKE4 (WDL4) protein plays a positive role in apical hook opening. WDL4 exhibits a temporal expression pattern during hook development in dark-grown seedlings that is directly regulated by ethylene signaling. WDL4 mutants showed a delayed hook opening phenotype while overexpression of WDL4 resulted in enhanced hook opening. In particular, wdl4-1 mutants exhibited stronger auxin accumulation in the concave side of the apical hook. Furthermore, the regulation of the auxin maxima and trafficking of the auxin efflux carriers PIN-FORMED1 (PIN1) and PIN7 in the hook region is critical for WDL4-mediated hook opening. Together, our study demonstrates that WDL4 positively regulates apical hook opening by modulating auxin distribution, thus unraveling a mechanism for MAP-mediated differential plant cell growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Ácidos Indolacéticos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Asociadas a Microtúbulos/genética , Células Vegetales , Plantas Modificadas Genéticamente , Transporte de Proteínas , Plantones/fisiología , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Plant growth is significantly altered in response to submergence stress. However, the molecular mechanisms used by seedlings in response to this stress, especially for hypocotyl growth, are largely unknown in terrestrial plants such as Arabidopsis thaliana. The microtubule cytoskeleton participates in plant cell growth, but it remains unclear whether submergence-mediated plant growth involves the microtubule cytoskeleton. We demonstrated that in Arabidopsis submergence induced underwater hypocotyl elongation through the activation of ethylene signaling, which modulated cortical microtubule reorganization. Submergence enhanced ethylene signaling, which then activated and stabilized its downstream transcription factor, phytochrome-interacting factor 3 (PIF3), to promote hypocotyl elongation. In particular, the regulation of microtubule organization was important for this physiological process. Microtubule-destabilizing protein 60 (MDP60), which was previously identified as a downstream effector of PIF3, played a positive role in submergence-induced hypocotyl elongation. Submergence induced MDP60 expression through ethylene signaling. The effects of submergence on hypocotyl elongation and cortical microtubule reorganization were suppressed in mdp60 mutants. These data suggest a potential mechanism by which submergence activates ethylene signaling to promote underwater hypocotyl elongation via alteration of the microtubule cytoskeleton in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Etilenos/metabolismo , Hipocótilo/crecimiento & desarrollo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Inmersión , Estrés FisiológicoRESUMEN
Plants perceive multiple physiological and environmental signals in order to fine-tune their growth and development. The highly dynamic plant cytoskeleton, including actin and microtubule networks, can rapidly alter their organization, stability and dynamics in response to internal and external stimuli, which is considered vital for plant growth and adaptation to the environment. The cytoskeleton-associated proteins have been shown to be key regulatory molecules in mediating cytoskeleton reorganization in response to multiple environmental signals, such as light, salt, drought and biotic stimuli. Recent findings, including our studies, have expanded knowledge about the functions and underlying mechanisms of the plant cytoskeleton in environmental adaptation.
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Citoesqueleto , Plantas , Actinas , Microtúbulos , Desarrollo de la PlantaRESUMEN
O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery and optimization of known bioactive scaffolds during drug development both demand efficient O-methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone polyketides. We show that rational, structure-guided active-site cavity engineering can reprogram the regioselectivity of these enzymes. We also characterize the interplay of engineered regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic tailoring of unnatural products toward the generation of diverse chemical matter for drug discovery and the PK/PD optimization of bioactive scaffolds for drug development.
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Lactonas/síntesis química , Metiltransferasas/química , Policétidos/síntesis química , Sustitución de Aminoácidos , Ascomicetos/enzimología , Dominio Catalítico , Humanos , Cinética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Ingeniería de Proteínas , Especificidad por Sustrato , Zearalenona/análogos & derivados , Zearalenona/metabolismo , Zeranol/análogos & derivados , Zeranol/metabolismoRESUMEN
Proper regulation of seed germination is essential for the successful propagation of a plant. The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) of the abscisic acid (ABA) signaling pathway plays a central role in the inhibition of seed germination. ABI5 is precisely regulated by the core ABA signaling components and multiple other factors. However, the complex regulatory network of ABI5 remains largely unknown. In this study, we determined the biochemical interaction between ABI5 and the BRINSENSITIVE1 (BRI1)-EMS-SUPPRESSOR1 (BES1) transcription factor of the brassinosteroid (BR) signaling pathway, as well as the function of BES1 regulating ABI5 during seed germination in Arabidopsis. BES1 directly interacts with ABI5 both in vitro and in vivo. The bZIP domain of ABI5, which is responsible for DNA binding, is critical for ABI5 binding to BES1. The interaction of BES1 with ABI5 significantly suppressed the binding of ABI5 to the promoter regions of downstream genes, which resulted in their reduced expression and consequently facilitated seed germination. This study shed new light on the coordination of multiple signaling pathways during seed germination. In particular, BES1 directly binds to ABI5, which interferes with its transcriptional activity and suppresses ABA signaling output.
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Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas de Unión al ADN/genética , Germinación , Semillas/genética , Semillas/fisiologíaRESUMEN
Regulation of cortical microtubule reorganization is essential for plant cell survival under high salinity conditions. In response to salt stress, microtubules undergo rapid depolymerization followed by reassembly to form a new microtubule network that promotes cell survival; however, the upstream regulatory mechanisms for this recovery response are largely unknown. In this study, we demonstrate that ethylene signaling facilitates salt stress-induced reassembly of cortical microtubules in Arabidopsis (Arabidopsis thaliana). Microtubule depolymerization was not affected under salt stress following the suppression of ethylene signaling with Ag+ or in ethylene-insensitive mutants, whereas microtubule reassembly was significantly inhibited. ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, was shown to play a central role in microtubule reassembly under salt stress. In addition, we performed functional characterization of the microtubule-stabilizing protein WAVE-DAMPENED2-LIKE5 (WDL5), which was found to promote ethylene-associated microtubule reassembly and plant salt stress tolerance. These findings indicate that ethylene signaling regulates microtubule reassembly by up-regulating WDL5 expression in response to salt stress, thereby implicating ethylene signaling in salt-stress tolerance in plants.
Asunto(s)
Arabidopsis/fisiología , Etilenos/metabolismo , Microtúbulos/metabolismo , Estrés Salino/fisiología , Transducción de Señal , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN , Técnicas de Inactivación de Genes , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Precise regulation of hypocotyl cell elongation is essential for plant growth and survival. Light suppresses hypocotyl elongation by degrading transcription factor phytochrome-interacting factor 3 (PIF3), whereas the phytohormone ethylene promotes hypocotyl elongation by activating PIF3. However, the underlying mechanisms regarding how these two pathways coordinate downstream effectors to mediate hypocotyl elongation are largely unclear. In this study, we identified the novel Microtubule-Destabilizing Protein 60 (MDP60), which plays a positive role in hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana); this effect is mediated through PIF3. Ethylene signaling up-regulates MDP60 expression via PIF3 binding to the MDP60 promoter. MDP60 loss-of-function mutants exhibit much shorter hypocotyls, whereas MDP60 overexpression significantly promotes hypocotyl cell elongation when grown in light compared to the control. MDP60 protein binds to microtubules in vitro and in vivo. The organization of cortical microtubules was significantly disrupted in mdp60 mutant cells and MDP60-overexpressing seedlings. These findings indicate that MDP60 is an important mediator of hypocotyl cell elongation. This study reveals a mechanism in which light and ethylene signaling coordinate MDP60 expression to modulate hypocotyl cell elongation by altering cortical microtubules in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Etilenos/farmacología , Hipocótilo/citología , Luz , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/efectos de los fármacos , Hipocótilo/crecimiento & desarrollo , Hipocótilo/efectos de la radiación , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/efectos de los fármacos , Microtúbulos/efectos de la radiación , Modelos Biológicos , Epidermis de la Planta/citología , Plantas Modificadas Genéticamente , Unión Proteica/efectos de los fármacos , Unión Proteica/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a well-known E3 ubiquitin ligase, functions as a central regulator of plant growth and photomorphogenic development in plants, including hypocotyl elongation. It has been well-established that, in darkness, COP1 targets many photomorphogenesis-promoting factors for ubiquitination and degradation in the nucleus. However, increasing evidence has shown that a proportion of COP1 is also localized outside the nucleus in dark-grown seedlings, but the physiological function of this localization remains largely unclear. In this study, we demonstrate that COP1 directly targets and mediates the degradation of WAVE-DAMPENED 2-LIKE 3 (WDL3) protein, a member of the microtubule-associated protein (MAP) WVD2/WDL family involved in regulating hypocotyl cell elongation of Arabidopsis seedlings. We show that COP1 interacts with WDL3 in vivo in a dark-dependent manner at cortical microtubules. Moreover, our data indicate that COP1 directly ubiquitinates WDL3 in vitro and that WDL3 protein is degraded in WT seedlings but is abundant in the cop1 mutant in the dark. Consistently, introduction of the wdl3 mutation weakened, whereas overexpression of WDL3 enhanced, the short-hypocotyl phenotype of cop1 mutant in darkness. Together, this study reveals a function of COP1 in regulating the protein turnover of a cytosol-localized MAP in etiolated hypocotyls, thus providing insights into COP1-mediated degradation of downstream factors to control seedling photomorphogenesis.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hipocótilo/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Oscuridad , Regulación del Desarrollo de la Expresión Génica , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Hipocótilo/efectos de la radiación , Luz , Fototransducción , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Desarrollo de la Planta/genética , Proteolisis , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/efectos de la radiación , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co-localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site-specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild-type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild-type Arabidopsis. Thus, we conclude that the PM-associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity-dependent manner, rather than its subcellular translocation.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/fisiología , Microtúbulos/metabolismo , Fosfolipasa D/metabolismo , Termotolerancia , Membrana Celular/metabolismo , Dinitrobencenos/farmacología , Prueba de Complementación Genética , Respuesta al Choque Térmico , Hipocótilo/citología , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Modelos Biológicos , Mutación/genética , Fenotipo , Polimerizacion , Unión Proteica , Fracciones Subcelulares/metabolismo , Sulfanilamidas/farmacologíaRESUMEN
Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN-DEPOLYMERIZING FACTORs (ADFs) typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs (ADF5 and ADF9) evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neofunctionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B (LatB) treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochemical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Tubo Polínico/metabolismo , Tubo Polínico/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Germinación/genética , Germinación/fisiología , Tubo Polínico/genéticaRESUMEN
How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.