RESUMO
Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Tubo Polínico/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , DNA Bacteriano , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutagênese Insercional , Infertilidade das Plantas , Tubo Polínico/enzimologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Ca(2+) is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca(2+) pumps conferred the ability to grow on Ca(2+)-depleted medium and tolerance to toxic levels of Mn(2+). A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca(2+) and Mn(2+) loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca(2+), indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 microm Mn(2+), indicating that AtECA3 is also important for the detoxification of excess Mn(2+). Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca(2+)-stimulated root growth and the detoxification of high Mn(2+), possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Endossomos/química , Manganês/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/química , Transporte Biológico , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/química , Glucuronidase/análise , Proteínas de Fluorescência Verde/análise , Dados de Sequência Molecular , Mutagênese Insercional , Peroxidases/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Alinhamento de Sequência , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
The Arabidopsis (Arabidopsis thaliana) compact inflorescence (cif) genotype causes altered adult vegetative development and a reduction in elongation of inflorescence internodes resulting in formation of floral clusters. The cif trait requires both a recessive mutation, cif1, and the activity of a naturally occurring dominant allele of an unlinked gene, CIF2(D). We show here that the pseudoverticillata mutation is allelic with cif1 and that the product of the CIF1 gene is ACA10, a member of the large family of P-type Ca(2+)-ATPases found in higher plants. T-DNA insertion mutations in ACA10, but not in the two other Arabidopsis plasma membrane Ca(2+)-ATPase-encoding genes, ACA8 and ACA9, cause a cif phenotype when combined with the dominant CIF2(D) modifier allele. Therefore, ACA10 has a unique function in regulating adult phase growth and inflorescence development. The wild-type ACA8 and ACA10 mRNAs are present at similar levels, and the two promoter-beta-glucuronidase fusion transgenes show very similar expression patterns. Moreover, transformation of the cif mutant with an extra copy of the ACA8 gene, which causes overexpression of the ACA8 transcript, can complement the cif phenotype. This suggests that these two Ca(2+) pump genes have distinct but related activities and that their differential functions can be altered by relatively small changes in their patterns or levels of expression. The correspondence between cif1 and mutations in ACA10 establishes a genetic link between calcium transport, vegetative phase change, and inflorescence architecture.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Flores/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/enzimologia , Alelos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ATPases Transportadoras de Cálcio/genética , DNA Bacteriano/genética , DNA de Plantas/genética , Flores/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Mutação , Filogenia , Folhas de Planta/genética , Plantas Geneticamente ModificadasRESUMO
Ion signals are critical to regulating polarized growth in many cell types, including pollen in plants and neurons in animals. Genetic evidence presented here indicates that pollen tube growth requires cyclic nucleotide-gated channel (CNGC) 18. CNGCs are nonspecific cation channels found in plants and animals and have well established functions in excitatory signal transduction events in animals. In Arabidopsis, male sterility was observed for two cngc18 null mutations. CNGC18 is expressed primarily in pollen, as indicated from a promoter::GUS (beta-glucuronidase) reporter analysis and expression profiling. The underlying cause of sterility was identified as a defect in pollen tube growth, resulting in tubes that were kinky, short, often thin, and unable to grow into the transmitting tract. Expression of a GFP-tagged CNGC18 in mutant pollen provided complementation and evidence for asymmetric localization of CNGC18 to the plasma membrane at the growing tip, starting at the time of pollen grain germination. Heterologous expression of CNGC18 in Escherichia coli resulted in a time- and concentration-dependent accumulation of more Ca2+. Thus, CNGC18 provides a mechanism to directly transduce a cyclic nucleotide (cNMP) signal into an ion flux that can produce a localized signal capable of regulating the pollen tip-growth machinery. These results identify a CNGC that is essential to an organism's life cycle and raise the possibility that CNGCs have a widespread role in regulating cell-growth dynamics in both plant and animals.
Assuntos
Polaridade Celular , Canais Iônicos/metabolismo , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ativação do Canal Iônico , Canais Iônicos/classificação , Canais Iônicos/genética , Pólen/citologiaRESUMO
STUDY DESIGN: Survey/case series. OBJECTIVE: To survey pain physicians about neurologic infarctions following cervical transforaminal epidural steroid injections (TF-ESIs). SUMMARY OF BACKGROUND DATA: Cervical TF-ESIs are commonly performed in patients with cervical radiculopathy, although there are no randomized controlled studies supporting their efficacy. Eight case reports of brain and spinal cord infarction have been published. In addition, one of the investigators (M.S.W.) has reviewed 4 cases of major cerebellum/brainstem infarction following cervical TF-ESIs with methylprednisolone. METHODS: To better characterize these complications, anonymous surveys were sent to all U.S. physician members of the American Pain Society. Respondents were asked about awareness of complications, year of occurrence, practice setting and specialty of the treating physician, use of fluoroscopy/contrast/local anesthetic/corticosteroid, doses administered, and CT/MRI/autopsy findings. RESULTS: Overall response rate was 21.4% (287 of 1340). In all, 78 complications were reported, including 16 vertebrobasilar brain infarcts, 12 cervical spinal cord infarcts, and 2 combined brain/spinal cord infarcts. Brain infarcts invariably involved the cerebellum, brainstem, or posterior cerebral artery territory. Thirteen cases resulted in a fatal outcome: 5 with brain infarcts, 1 with combined brain/spinal cord infarcts, 1 following high spinal anesthesia, 1 associated with a seizure, and 5 with unspecified etiology. All 4 cases with corticosteroid alone involved methylprednisolone, resulting in 3 cerebellar infarcts and 1 posterior cerebral territory infarct. Of these, 3 had fatal outcomes and 2 autopsies revealed no vertebral artery trauma. CONCLUSIONS: This study demonstrates a significant risk of serious neurologic injury after cervical TF-ESIs. A growing body of evidence supports an embolic mechanism, whereby inadvertent intra-arterial injection of particulate corticosteroid causes a distal infarct. Embolism to the distal basilar artery region can cause midbrain, pons, cerebellum, thalamus, temporal and occipital lobe infarctions. Other potential mechanisms of infarction include vertebral artery perforation causing dissection/thrombosis and needle-induced vasospasm.
Assuntos
Anti-Inflamatórios/efeitos adversos , Infarto Encefálico/etiologia , Vértebras Cervicais , Injeções Epidurais/efeitos adversos , Traumatismos da Medula Espinal/etiologia , Corticosteroides/efeitos adversos , Anti-Inflamatórios/administração & dosagem , Infarto Encefálico/induzido quimicamente , Infarto Encefálico/mortalidade , Estudos Transversais , Humanos , Injeções Epidurais/estatística & dados numéricos , Metilprednisolona/efeitos adversos , Radiculopatia/tratamento farmacológico , Fatores de Risco , Traumatismos da Medula Espinal/induzido quimicamente , Traumatismos da Medula Espinal/mortalidade , Inquéritos e Questionários , Estados Unidos/epidemiologiaRESUMO
Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Pólen/crescimento & desenvolvimento , Arabidopsis/genética , Sinalização do Cálcio/fisiologia , Fertilização , Genoma de Planta , Plasmídeos , Pólen/enzimologia , Pólen/fisiologiaRESUMO
Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.