RESUMO
Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2 effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P2 in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2 functions to coordinate cytoskeletal dynamics and secretion.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Nicotiana/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/genética , Regulação da Expressão Gênica de Plantas , Tubo Polínico/genética , Tubo Polínico/metabolismo , Nicotiana/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP. Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted. When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2 -dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant-oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM. In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2 -dependent processes during plant immunity.
Assuntos
Phytophthora infestans , Solanum tuberosum , Moléculas com Motivos Associados a Patógenos , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases , Doenças das PlantasRESUMO
An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.
Assuntos
Arabidopsis/enzimologia , Membrana Celular/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotiana/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Tubo Polínico/enzimologia , Tubo Polínico/crescimento & desenvolvimento , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/citologia , Biocatálise , Endocitose , Corantes Fluorescentes/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Modelos Biológicos , Fosforilação , Fosfotreonina/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Tubo Polínico/citologia , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes , Nicotiana/citologiaRESUMO
The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Moléculas com Motivos Associados a Patógenos/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relação Dose-Resposta a Droga , Flagelina/química , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Moléculas com Motivos Associados a Patógenos/administração & dosagem , Moléculas com Motivos Associados a Patógenos/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Protoplastos/metabolismoRESUMO
The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.
Assuntos
Arabidopsis/metabolismo , Transporte Biológico , Vesículas Revestidas por Clatrina/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Polaridade Celular , Endocitose , Proteínas de Fluorescência Verde/análise , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Sementes/embriologia , Sementes/metabolismo , Solanum lycopersicum/embriologia , Solanum lycopersicum/metabolismo , Apoptose/efeitos dos fármacos , Ciclopentanos/farmacologia , Endosperma/efeitos dos fármacos , Endosperma/metabolismo , Frutas/efeitos dos fármacos , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Óvulo Vegetal/efeitos dos fármacos , Óvulo Vegetal/enzimologia , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA/efeitos dos fármacos , Sementes/efeitos dos fármacosRESUMO
The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Nicotiana/crescimento & desenvolvimento , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/metabolismo , DNA Bacteriano/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Mutagênese Insercional , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Tubo Polínico/genética , Tubo Polínico/metabolismo , Tiazolidinas/farmacologia , Nicotiana/genética , Nicotiana/metabolismo , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Ciclopentanos/metabolismo , Oxirredutases Intramoleculares/genética , Oxilipinas/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dimerização , Flores/citologia , Flores/efeitos dos fármacos , Flores/enzimologia , Flores/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes Reporter , Oxirredutases Intramoleculares/metabolismo , Família Multigênica , Mutação , Especificidade de Órgãos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Feixe Vascular de Plantas/citologia , Feixe Vascular de Plantas/efeitos dos fármacos , Feixe Vascular de Plantas/enzimologia , Feixe Vascular de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , RNA Mensageiro/genética , RNA de Plantas/genética , Sementes/citologia , Sementes/efeitos dos fármacos , Sementes/enzimologia , Sementes/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Lipid homeostasis allows cells to adjust membrane biophysical properties in response to changes in environmental conditions. In the yeast Saccharomyces cerevisiae, a downward shift in temperature from an optimal reduces membrane fluidity, which triggers a lipid remodeling of the plasma membrane. How changes in membrane fluidity are perceived, and how the abundance and composition of different lipid classes is properly balanced, remain largely unknown. Here, we show that the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the most abundant plasma membrane phosphoinositide, drop rapidly in response to a downward shift in temperature. This change triggers a signaling cascade transmitted to cytosolic diphosphoinositol phosphate derivatives, among them 5-PP-IP4 and 1-IP7, that exert regulatory functions on genes involved in the inositol and phospholipids (PLs) metabolism, and inhibit the activity of the protein kinase Pho85. Consistent with this, cold exposure triggers a specific program of neutral lipids and PLs changes. Furthermore, we identified Pho85 as playing a key role in controlling the synthesis of long-chain bases (LCBs) via the Ypk1-Orm2 regulatory circuit. We conclude that Pho85 orchestrates a coordinated response of lipid metabolic pathways that ensure yeast thermal adaptation.
Assuntos
Aclimatação/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Membrana Celular/metabolismo , Temperatura Baixa/efeitos adversos , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Fluidez de Membrana/fisiologia , Redes e Vias Metabólicas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P2-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P2 antagonizes the effects of PtdIns(4,5)P2 overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the subapical plasma membrane of pollen tubes.
RESUMO
Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Resistência à Doença/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transcriptoma/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Parede Celular/química , Parede Celular/metabolismo , Endossomos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Plasmodioforídeos/patogenicidade , Via Secretória/genética , Solo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Plants exposed to hyperosmotic stress undergo changes in membrane dynamics and lipid composition to maintain cellular integrity and avoid membrane leakage. Various plant species respond to hyperosmotic stress with transient increases in PtdIns(4,5)P(2); however, the physiological role of such increases is unresolved. The plasma membrane represents the outermost barrier between the symplast of plant cells and its apoplastic surroundings. In the present study, the spatio-temporal dynamics of stress-induced changes in phosphoinositides were analysed in subcellular fractions of Arabidopsis leaves to delineate possible physiological roles. Unlabelled lipids were separated by TLC and quantified by gas-chromatographic detection of associated fatty acids. Transient PtdIns(4,5)P(2) increases upon exposure to hyperosmotic stress were detected first in enriched plasmamembrane fractions, however, at later time points, PtdIns(4,5)P(2) was increased in the endomembrane fractions of the corresponding two-phase systems. When major endomembranes were enriched from rosette leaves prior to hyperosmotic stress and during stimulation for 60 min, no stress-induced increases in the levels of PtdIns(4,5)P(2) were found in fractions enriched for endoplasmic reticulum, nuclei or plastidial membranes. Instead, increased PtdIns(4,5)P(2) was found in CCVs (clathrin-coated vesicles), which proliferated several-fold in mass within 60 min of hyperosmotic stress, according to the abundance of CCV-associated proteins and lipids. Monitoring the subcellular distribution of fluorescence-tagged reporters for clathrin and PtdIns(4,5)P(2) during transient co-expression in onion epidermal cells indicates rapid stress-induced co-localization of clathrin with PtdIns(4,5)P(2) at the plasma membrane. The results indicate that PtdIns(4,5)P(2) may act in stress-induced formation of CCVs in plant cells, highlighting the evolutionary conservation of the phosphoinositide system between organismic kingdoms.
Assuntos
Arabidopsis/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Pressão Osmótica , Fosfatidilinositol 4,5-Difosfato/análise , Folhas de Planta/metabolismoRESUMO
The allene oxide cyclase (AOC) catalyzes the formation of cis-(+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the beta-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.
Assuntos
Regulação da Expressão Gênica de Plantas , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Regiões Promotoras Genéticas/genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/crescimento & desenvolvimento , Clonagem Molecular , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Solanum lycopersicum/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimentoRESUMO
Eukaryotic membranes contain small amounts of phospholipids that have regulatory effects on the physiological functions of cells, tissues, and organs. Phosphoinositides (PIs)-the phosphorylated derivatives of phosphatidylinositol-are one example of such regulatory lipids. Although PIs were described in plants decades ago, their contribution to the regulation of physiological processes in plants is not well understood. In the past few years, evidence has emerged that PIs are essential for plant function and development. Recently reported phenotypes associated with the perturbation of different PIs suggest that some subgroups of PIs influence specific processes. Although the molecular targets of PI-dependent regulation in plants are largely unknown, the effects of perturbed PI metabolism can be used to propose regulatory modules that involve particular downstream targets of PI regulation. This review summarizes phenotypes associated with the perturbation of the plant PI network to categorize functions and suggest possible downstream targets of plant PI regulation.
Assuntos
Fosfatidilinositóis/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transporte Biológico , Modelos Biológicos , Fenótipo , Fosfatidilinositóis/química , Fosfatidilinositóis/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Plantas/ultraestruturaRESUMO
Plants respond to mechanical wounding or herbivore attack with a complex scenario of sequential, antagonistic or synergistic action of different signals leading to defense gene expression. Tomato plants were used as a model system since the peptide systemin and the lipid-derived jasmonic acid (JA) were recognized as essential signals in wound-induced gene expression. In this review recent data are discussed with emphasis on wound-signaling in tomato. The following aspects are covered: (i) systemin signaling, (ii) JA biosynthesis and action, (iii) orchestration of various signals such as JA, H2O2, NO, and salicylate, (iv) local and systemic response, and (v) amplification in wound signaling. The common occurrence of JA biosynthesis and systemin generation in the vascular bundles suggest JA as the systemic signal. Grafting experiments with JA-deficient, JA-insensitive and systemin-insensitive mutants strongly support this assumption.
Assuntos
Ciclopentanos/metabolismo , Solanum lycopersicum/metabolismo , Peróxido de Hidrogênio/metabolismo , Imunidade Inata , Solanum lycopersicum/citologia , Solanum lycopersicum/crescimento & desenvolvimento , Modelos Biológicos , Óxido Nítrico/metabolismo , Oxilipinas , Peptídeos/metabolismo , Salicilatos/metabolismo , Transdução de SinaisRESUMO
The peroxidation of polyunsaturated fatty acids, common to all eukaryotes, is mostly catalyzed by members of the lipoxygenase enzyme family of non-heme iron containing dioxygenases. Lipoxygenase products can be metabolized further in the oxylipin pathway by several groups of CYP74 enzymes. One prominent oxylipin is jasmonic acid (JA), a product of the 13-allene oxide synthase branch of the pathway and known as signaling substance that plays a role in vegetative and propagative plant development as well as in plant responses to wounding and pathogen attack. In barley roots, JA level increases upon colonization by arbuscular mycorrhizal fungi. Apart from this first result regarding JA, no information is available on the relevance of lipidperoxide metabolism in arbuscular mycorrhizal symbiosis. Thus we analyzed fatty acid and lipidperoxide patterns in roots of Medicago truncatula during mycorrhizal colonization. Levels of fungus-specific fatty acids as well as palmitic acid (16:0) and oleic acid (18:1 n - 9) were increased in mycorrhizal roots. Thus the degree of arbuscular mycorrhizal colonization of roots can be estimated via analysis of fungal specific esterified fatty acids. Otherwise, no significant changes were found in the profiles of esterified and free fatty acids. The 9- and 13-LOX products of linoleic and alpha-linolenic acid were present in all root samples, but did not show significant differences between mycorrhizal and non-mycorrhizal roots, except JA which showed elevated levels in mycorrhizal roots. In both types of roots levels of 13-LOX products were higher than those of 9-LOX products. In addition, three cDNAs encoding CYP74 enzymes, two 9/13-hydroperoxide lyases and a 13-allene oxide synthase, were isolated and characterized. The transcript accumulation of these three genes, however, was not increased in mycorrhizal roots of M. truncatula.
Assuntos
Metabolismo dos Lipídeos , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Micorrizas/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Peroxidação de Lipídeos/fisiologia , Lipídeos/química , Estrutura Molecular , Micorrizas/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Simbiose/fisiologia , Fatores de TempoRESUMO
Jasmonic acid (JA) and its derivatives are well-characterized signaling molecules in plant defense and development, but the site of their localization within plant tissue is entirely unknown. To address the question whether applied JA accumulates extracellularly or intracellularly, leaves of tomato and barley were fed with 14C-labeled JA and the label was localized in cryofixed and lyophilized leaf tissues by microautoradiography. In tomato the radioactivity was detectable within the apoplast, but no label was found within the mesophyll cells. By contrast, in barley leaf tissues, radioactivity was detected within the mesophyll cells suggesting a cellular uptake of exogenously applied JA. JA, applied to leaves of both plants as in the labeling experiments, led in all leaf cells to the expression of JA-inducible genes indicating that the perception is completed by JA signal transduction.
Assuntos
Ciclopentanos/farmacologia , Hordeum/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Solanum lycopersicum/efeitos dos fármacos , Autorradiografia , Radioisótopos de Carbono/metabolismo , Ciclopentanos/administração & dosagem , Regulação da Expressão Gênica de Plantas , Hordeum/citologia , Hibridização In Situ , Solanum lycopersicum/citologia , Oxilipinas , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Coronalon, a synthetic 6-ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12-oxo-phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense-related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress-related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.
Assuntos
Isoleucina/análogos & derivados , Isoleucina/farmacologia , Fenômenos Fisiológicos Vegetais , Plantas/efeitos dos fármacos , Células Cultivadas , Ciclopentanos/classificação , Ciclopentanos/farmacologia , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados/farmacologia , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Isoleucina/química , Cinética , Estrutura Molecular , Oxilipinas , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Relação Estrutura-AtividadeRESUMO
Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.