ABSTRACT
Because of the large amount of energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system, and the developmental coordination of these organs under conditions of reduced nitrogen (N) availability remains elusive. We show that the Medicago truncatula COMPACT ROOT ARCHITECTURE2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low N. C-TERMINALLY ENCODED PEPTIDE (MtCEP1) peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of YUCCA2 (MtYUC2) auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by overexpressing MtYUC2, and conversely, a treatment with YUC inhibitors or an MtYUC2 knockout rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and thereby repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement with this interaction, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.
Subject(s)
Ethylenes/metabolism , Indoleacetic Acids/metabolism , Medicago truncatula/metabolism , Plant Proteins/metabolism , Plant Root Nodulation/physiology , Gene Expression Regulation, Plant , Medicago truncatula/growth & development , Mutation , Phosphorylation , Plant Proteins/genetics , Plant Roots/physiology , Plant Shoots/genetics , Plants, Genetically Modified , Protein Kinases/genetics , Protein Kinases/metabolism , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Rhizobium/physiology , Serine/metabolism , SymbiosisABSTRACT
Because of the high energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system and the developmental coordination of these organs according to reduced nitrogen (N) availability remains elusive. We show that the Compact Root Architecture 2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low-N. MtCEP1 peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of MtYUC2 auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by over-expressing MtYUC2, and conversely, a treatment with YUC inhibitors or a MtYUC2 knock-out rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and therefore repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.
ABSTRACT
Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.
Subject(s)
Peptides/chemistry , Plant Root Nodulation/physiology , Rhizobium/metabolism , Transcription Factors/metabolism , Cytokinins/metabolism , Epidermis , Gene Expression Regulation, Plant , Lotus/metabolism , Medicago truncatula , Peptides/genetics , Plant Proteins , Plant Root Nodulation/genetics , Plant Roots/metabolism , Promoter Regions, Genetic , Protein Kinases , Protein Sorting Signals/genetics , Root Nodules, Plant , Sinorhizobium meliloti/metabolism , SymbiosisABSTRACT
Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.
Subject(s)
Cytokinins/metabolism , Plant Root Nodulation , Signal Transduction , Transcription Factors/metabolism , Cell Nucleus Size , Endoreduplication , Gene Expression Regulation, Plant , Genes, Plant , Medicago truncatula/genetics , Medicago truncatula/microbiology , Phenotype , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Root Nodulation/genetics , Promoter Regions, Genetic , Protein Binding , Protein Domains , Sinorhizobium meliloti/physiology , Transcriptional Activation/geneticsABSTRACT
BACKGROUND: Legumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively. RESULTS: A striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this "core" module potentially acts in most plant organs including nodules. CONCLUSIONS: Further functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.
Subject(s)
Cytokinins/metabolism , Histidine Kinase/genetics , Medicago truncatula/genetics , Transcription Factors/genetics , Evolution, Molecular , Fabaceae/genetics , Fabaceae/metabolism , Gene Expression Regulation, Plant , Histidine Kinase/metabolism , Medicago truncatula/metabolism , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Root Nodules, Plant/metabolism , Signal Transduction , Transcription Factors/metabolism , Whole Genome SequencingABSTRACT
In legume plants, low-nitrogen soils promote symbiotic interactions with rhizobial bacteria, leading to the formation of nitrogen-fixing root nodules. Among critical signals regulating this developmental process are bacterial Nod Factors (NFs) and several plant hormones, including cytokinins (CKs) and gibberellins (GAs). Here, we show in Medicago truncatula that GA signaling mediated by DELLA1 decreases the amount of bioactive CKs in roots and negatively impacts the Cytokinin Response1 (CRE1)-dependent NF activation of a subset of CK-signaling genes as well as of the CK-regulated Nodulation Signaling Pathway2 and Ethylene Response Factor Required for Nodulation1 early nodulation genes. Consistently, a dominant-active DELLA1 protein can partially rescue the reduced nodulation of the cre1 mutant and triggers the formation of nodule-like structures when expressed in the root cortex or in the root epidermis. This suggests a model where the DELLA1-mediated GA signaling interplays with the CRE1-dependent CK pathway to regulate early nodulation in response to both NF and CK signals critical for this symbiotic interaction.
Subject(s)
Gene Expression Regulation, Plant/physiology , Gibberellins/metabolism , Medicago truncatula/metabolism , Plant Proteins/metabolism , Plant Root Nodulation/physiology , Medicago truncatula/genetics , Plant Proteins/genetics , Plant Roots , Root Nodules, Plant/microbiology , Signal Transduction , Sinorhizobium meliloti/physiology , Symbiosis , Transcription FactorsABSTRACT
Substantial progress has been made in the understanding of early stages of the symbiotic interaction between legume plants and rhizobium bacteria. Those include the specific recognition of symbiotic partners, the initiation of bacterial infection in root hair cells, and the inception of a specific organ in the root cortex, the nodule. Increasingly complex regulatory networks have been uncovered in which cytokinin (CK) phytohormones play essential roles in different aspects of early symbiotic stages. Intriguingly, these roles can be either positive or negative, cell autonomous or non-cell autonomous, and vary, depending on time, root tissues, and possibly legume species. Recent developments on CK symbiotic functions and interconnections with other signaling pathways during nodule initiation are the focus of this review.
Subject(s)
Cytokinins/physiology , Plant Root Nodulation/physiology , Cytokinins/genetics , Fabaceae/microbiology , Fabaceae/physiology , Nitrogen Fixation/physiology , Plant Root Nodulation/genetics , Plant Roots/microbiology , Plant Roots/physiology , Rhizobium/physiology , Symbiosis/physiologyABSTRACT
Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections.
Subject(s)
Gibberellins/metabolism , Medicago truncatula/microbiology , Plant Root Nodulation/physiology , Root Nodules, Plant/microbiology , Sinorhizobium meliloti/metabolism , CCAAT-Binding Factor/metabolism , Lipopolysaccharides/metabolism , Medicago truncatula/genetics , Plant Proteins/biosynthesis , Plant Proteins/metabolism , Signal Transduction , Sinorhizobium meliloti/growth & developmentABSTRACT
Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE-domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock-down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume-specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation.
Subject(s)
Medicago truncatula/microbiology , Receptors, Cell Surface/physiology , Root Nodules, Plant/growth & development , Arabidopsis/genetics , Cytokinins/metabolism , Genome, Plant , Medicago truncatula/metabolism , Nitrogen Fixation , Plant Growth Regulators/metabolism , Plant Growth Regulators/physiology , Plants, Genetically Modified/microbiology , Receptors, Cell Surface/metabolism , Root Nodules, Plant/genetics , Root Nodules, Plant/metabolism , Signal Transduction , Sinorhizobium/physiology , SymbiosisABSTRACT
Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:ß-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.
Subject(s)
Cytokinins/metabolism , Lipopolysaccharides/metabolism , Medicago truncatula/metabolism , Plant Epidermis/metabolism , Plant Roots/metabolism , Gene Expression Regulation, Plant , Lasers , Lipopolysaccharides/pharmacology , Medicago truncatula/genetics , Plant Epidermis/drug effects , Plant Epidermis/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plants, Genetically Modified , Root Nodules, Plant/genetics , Root Nodules, Plant/metabolism , Sequence Analysis, RNA/methods , Signal TransductionABSTRACT
Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.
Subject(s)
Cytokinins/pharmacology , Medicago truncatula/physiology , Plant Growth Regulators/pharmacology , Plant Root Nodulation/genetics , Sinorhizobium meliloti/physiology , Transcription Factors/metabolism , Amino Acid Sequence , Consensus Sequence , Gene Expression Profiling , Gene Expression Regulation, Plant , Medicago truncatula/drug effects , Medicago truncatula/genetics , Medicago truncatula/microbiology , MicroRNAs/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Nitrogen Fixation , Oligonucleotide Array Sequence Analysis , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Promoter Regions, Genetic/genetics , Root Nodules, Plant/drug effects , Root Nodules, Plant/genetics , Root Nodules, Plant/microbiology , Root Nodules, Plant/physiology , Seedlings/drug effects , Seedlings/genetics , Seedlings/microbiology , Seedlings/physiology , Sequence Alignment , Signal Transduction , Symbiosis , Transcription Factors/genetics , TranscriptomeABSTRACT
Snf1-related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA-independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site-directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two-dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA-responsiveness.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Abscisic Acid/pharmacology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Binding Sites/genetics , Biocatalysis/drug effects , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Enzyme Activation/drug effects , Isoenzymes/genetics , Isoenzymes/metabolism , Mass Spectrometry , Mutagenesis, Site-Directed , Osmolar Concentration , Phosphorylation , Plant Growth Regulators/pharmacology , Protein Binding , Protein Kinases/genetics , Protein Serine-Threonine Kinases/geneticsABSTRACT
In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.
Subject(s)
Abscisic Acid/pharmacology , Anion Transport Proteins/metabolism , Arabidopsis/drug effects , Cell Membrane/drug effects , Proton Pumps/metabolism , Reactive Oxygen Species/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Calcium Signaling , Cell Membrane/physiology , Cells, Cultured , Electrophysiology , Gene Expression Regulation, Plant , Plant Growth Regulators/pharmacology , RNA, Plant/geneticsABSTRACT
Brassinosteroids (BRs) are involved in numerous physiological processes associated with plant development and especially with cell expansion. Here we report that two BRs, 28-homobrassinolide (HBL) and its direct precursor 28-homocastasterone (HCS), promote cell expansion of Arabidopsis thaliana suspension cells. We also show that cell expansions induced by HBL and HCS are correlated with the amplitude of the plasma membrane hyperpolarization they elicited. HBL, which promoted the larger cell expansion, also provoked the larger hyperpolarization. We observed that membrane hyperpolarization and cell expansion were partially inhibited by the proton pump inhibitor erythrosin B, suggesting that proton pumps were not the only ion transport system modulated by the two BRs. We used a voltage clamp approach in order to find the other ion transport systems involved in the PM hyperpolarization elicited by HBL and HCS. Interestingly, while anion currents were inhibited by both HBL and HCS, outward rectifying K+ currents were increased by HBL but inhibited by HCS. The different electrophysiological behavior shown by HBL and HCS indicates that small changes in the BR skeleton might be responsible for changes in bioactivity.
Subject(s)
Anions/metabolism , Arabidopsis/metabolism , Ion Channels/metabolism , Proton Pumps/metabolism , Steroids/physiology , Arabidopsis/cytology , Cell Membrane/metabolismABSTRACT
Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted harpin, HrpN ea, is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. We show here that HrpN ea induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis transmembrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN ea-induced cell death.
Subject(s)
Arabidopsis/physiology , Bacterial Outer Membrane Proteins/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/agonists , Erwinia amylovora/metabolism , Arabidopsis/cytology , Arabidopsis/drug effects , Bacterial Outer Membrane Proteins/pharmacology , Cell Culture Techniques , Cell Death , Glyburide/pharmacology , Ion Channel Gating , Tetramisole/pharmacologyABSTRACT
In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis/metabolism , Diphosphates/metabolism , Glycerol/analogs & derivatives , Glycerol/metabolism , Second Messenger Systems , Arabidopsis Proteins/metabolism , Cells, Cultured , Gene Expression Regulation, Plant/physiology , Phosphatidic Acids/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca(2+)](cyt) enhanced anion channels and impaired H(+)-ATPases.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis/metabolism , Calcium/metabolism , Cell Membrane/metabolism , Ion Channels/metabolism , Proton Pumps/metabolism , Arabidopsis/cytology , Arabidopsis/drug effects , Calcium Signaling/drug effects , Cell Membrane/drug effects , Cells, Cultured , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Plant Growth Regulators/pharmacology , Proton Pump Inhibitors , Proton-Translocating ATPases/antagonists & inhibitors , Proton-Translocating ATPases/metabolismABSTRACT
Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cell Membrane/physiology , Phospholipase D/metabolism , rab GTP-Binding Proteins/genetics , Arabidopsis/cytology , Arabidopsis/enzymology , Cell Membrane/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Heterotrimeric GTP-Binding Proteins/metabolism , Ion Channels/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Substrate Specificity , Type C Phospholipases/metabolismABSTRACT
Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.