RESUMEN
Aluminum-dependent stoppage of root growth requires the DNA damage response (DDR) pathway including the p53-like transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), which promotes terminal differentiation of the root tip in response to Al dependent cell death. Transcriptomic analyses identified Al-induced SOG1-regulated targets as candidate mediators of this growth arrest. Analysis of these factors either as loss-of-function mutants or by overexpression in the als3-1 background shows ERF115, which is a key transcription factor that in other scenarios is rate-limiting for damaged stem cell replenishment, instead participates in transition from an actively growing root to one that has terminally differentiated in response to Al toxicity. This is supported by a loss-of-function erf115 mutant raising the threshold of Al required to promote terminal differentiation of Al hypersensitive als3-1. Consistent with its key role in stoppage of root growth, a putative ERF115 barley ortholog is also upregulated following Al exposure, suggesting a conserved role for this ATR-dependent pathway in Al response. In contrast to other DNA damage agents, these results show that ERF115 and likely related family members are important determinants of terminal differentiation of the root tip following Al exposure and central outputs of the SOG1-mediated pathway in Al response.
RESUMEN
Aluminium (Al) ions are one of the primary growth-limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al-containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double-strand breaks. We find that the Al-induced damage specifically involves homology-dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi-level response to Al-induced DNA damage in plants.
Asunto(s)
Aluminio/toxicidad , Arabidopsis/genética , Daño del ADN/genética , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.
Asunto(s)
Aluminio/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN/efectos de los fármacos , Hordeum/efectos de los fármacos , Hidrazida Maleica/farmacología , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Daño del ADN/genética , Genoma de Planta/efectos de los fármacos , Genoma de Planta/genética , Genotipo , Hordeum/genética , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Mutágenos/farmacología , Mutación/efectos de los fármacos , Mutación/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genéticaRESUMEN
By screening for suppressors of the aluminum (Al) hypersensitive Arabidopsis thaliana mutant als3-1, it was found that mutational loss of the Arabidopsis DNA damage response transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) confers increased Al tolerance similar to the loss-of-function mutants for the cell cycle checkpoint genes ATAXIA TELANGIECTASIA AND RAD3 RELATED (ATR) and ALUMINUM TOLERANT2 (ALT2). This suggests that Al-dependent terminal differentiation of the root tip is an active process resulting from activation of the DNA damage checkpoint by an ATR-regulated pathway, which functions at least in part through SOG1. Consistent with this, ATR can phosphorylate SOG1 in vitro. Analysis of SOG1's role in Al-dependent root growth inhibition shows that sog1-7 prevents Al-dependent quiescent center differentiation and endoreduplication in the primary root tip. Following Al exposure, SOG1 increases expression of several genes previously associated with DNA damage, including BRCA1 and PARP2, with gel-shift analysis showing that SOG1 can physically associate with the BRCA1 promoter in vitro. Al-responsive expression of these SOG1-regulated genes requires ATR and ALT2, but not ATAXIA TELANGIECTASIA MUTATED, thus demonstrating that in response to chronic Al exposure, ATR, ALT2, and SOG1 function together to halt root growth and promote terminal differentiation at least in part in a transcription-dependent manner.
Asunto(s)
Aluminio/toxicidad , Proteínas de Arabidopsis/genética , Arabidopsis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Meristema/efectos de los fármacos , Factores de Transcripción/genética , Transportadoras de Casetes de Unión a ATP/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Meristema/citología , Meristema/genética , Mutación , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
A suppressor mutagenesis screen was conducted in order to identify second site mutations that could reverse the extreme hypersensitivity to aluminium (Al) seen for the Arabidopsis mutant, als3-1. From this screen, it was found that a loss-of-function mutation in the previously described SUV2 (SENSITIVE TO UV 2), which encodes a putative plant ATRIP homologue that is a component of the ATR-dependent cell checkpoint response, reversed the als3-1 phenotype. This included prevention of hallmarks associated with als3-1 including Al-dependent terminal differentiation of the root tip and transition to endoreduplication. From this analysis, SUV2 was determined to be required for halting cell cycle progression and triggering loss of the quiescent centre (QC) following exposure to Al. In conjunction with this, SUV2 was found to have a similar role as ATR, ALT2 and SOG1 in Al-dependent stoppage of root growth, all of which are required for promotion of expression of a suite of genes that likely are part of an Al-dependent DNA damage transcriptional response. This work argues that these Al response factors work together to detect Al-dependent damage and subsequently activate a DNA damage response pathway that halts the cell cycle and subsequently promotes QC differentiation and entrance into endocycling.
Asunto(s)
Aluminio/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas Portadoras/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Daño del ADN , ADN de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Supresores , Mutación/genéticaRESUMEN
Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Etilenos/farmacología , Mutación/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica de las Plantas , Genes Supresores , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Supresión Genética , Factores de Transcripción/metabolismoRESUMEN
Aluminum (Al) toxicity is a global issue that severely limits root growth in acidic soils. Isolation of suppressors of the Arabidopsis thaliana Al-hypersensitive mutant, als3-1, resulted in identification of a cell cycle checkpoint factor, ALUMINUM TOLERANT2 (ALT2), which monitors and responds to DNA damage. ALT2 is required for active stoppage of root growth after Al exposure, because alt2 loss-of-function mutants fail to halt root growth after Al exposure, do not accumulate CyclinB1;1 in the root tip, and fail to force differentiation of the quiescent center. Thus, alt2-1 mutants are highly tolerant of Al levels that are severely inhibitory to the wild type. The alt2-1 allele is a loss-of-function mutation in a protein containing a putative DDB1-binding WD40 motif, previously identified as TANMEI, which is required for assessment of DNA integrity, including monitoring of DNA crosslinks. alt2-1 and atr loss-of-function mutants, the latter of which affects the cell cycle checkpoint ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED, are severely sensitive to DNA crosslinking agents and have increased Al tolerance. These results suggest that Al likely acts as a DNA-damaging agent in vivo and that Al-dependent root growth inhibition, in part, arises from detection of and response to this damage by TANMEI/ALT2 and ATR, both of which actively halt cell cycle progression and force differentiation of the quiescent center.
Asunto(s)
Aluminio/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Clonación Molecular , Daño del ADN , Prueba de Complementación Genética , Raíces de Plantas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas R-SNARE/genética , Transducción de Señal/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/fisiología , Mutación , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Mapeo de Interacción de Proteínas , Proteínas R-SNARE/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Aluminum (Al) toxicity is a global problem severely limiting agricultural productivity in acid-soil regions comprising upwards of 50% of the world's arable land [1, 2]. Although Al-exclusion mechanisms have been intensively studied [3-9], little is known about tolerance to internalized Al, which is predicted to be mechanistically complex because of the plethora of predicted cellular targets for Al(3+)[2, 10]. An Arabidopsis mutant with Al hypersensitivity, als3-1, was found to represent a lesion in a phloem and root-tip-localized factor similar to the bacterial ABC transporter ybbm, with ALS3 likely responsible for Al transfer from roots to less-sensitive tissues [10-12]. To identify mutations that enhance mechanisms of Al resistance or tolerance, a suppressor screen for mutants that mask the Al hypersensitivity of als3-1 was performed [13]. Two allelic suppressors conferring increased Al tolerance were found to represent dominant-negative mutations in a factor required for monitoring DNA integrity, AtATR[14-17]. From this work, Al-dependent root-growth inhibition primarily arises from DNA damage coupled with AtATR-controlled blockage of cell-cycle progression and terminal differentiation because of loss of the root-quiescent center, with mutations that prevent response to this damage resulting in quiescent-center maintenance and sustained vigorous growth in an Al-toxic environment.
Asunto(s)
Aluminio/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Daño del ADN , Raíces de Plantas/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Aluminum (Al) toxicity is considered to be the most harmful abiotic stress in acidic soils that today comprise more than 50% of the world's arable lands. Barley belongs to a group of crops that are most sensitive to Al in low pH soils. We present the RNA-seq analysis of root meristems of barley seedlings grown in hydroponics at optimal pH (6.0), low pH (4.0), and low pH with Al (10 µM of bioavailable Al3+ ions). Two independent experiments were conducted: with short-term (24 h) and long-term (7 days) Al treatment. In the short-term experiment, more genes were differentially expressed (DEGs) between root meristems grown at pH = 6.0 and pH = 4.0, than between those grown at pH = 4.0 with and without Al treatment. The genes upregulated by low pH were associated mainly with response to oxidative stress, cell wall organization, and iron ion binding. Among genes upregulated by Al, overrepresented were those related to response to stress condition and calcium ion binding. In the long-term experiment, the number of DEGs between hydroponics at pH = 4.0 and 6.0 were lower than in the short-term experiment, which suggests that plants partially adapted to the low pH. Interestingly, 7 days Al treatment caused massive changes in the transcriptome profile. Over 4,000 genes were upregulated and almost 2,000 genes were downregulated by long-term Al stress. These DEGs were related to stress response, cell wall development and metal ion transport. Based on our results we can assume that both, Al3+ ions and low pH are harmful to barley plants. Additionally, we phenotyped the root system of barley seedlings grown in the same hydroponic conditions for 7 days at pH = 6.0, pH = 4.0, and pH = 4.0 with Al. The results correspond to transcriptomic data and show that low pH itself is a stress factor that causes a significant reduction of root growth and the addition of aluminum further increases this reduction. It should be noted that in acidic arable lands, plants are exposed simultaneously to both of these stresses. The presented transcriptome analysis may help to find potential targets for breeding barley plants that are more tolerant to such conditions.
RESUMEN
An Arabidopsis mutant, eer5-1, which has an enhanced ethylene response in etiolated seedlings, including hypersensitivity and extreme exaggeration of response to ethylene, was isolated and characterized. As with other identified eer mutants, the enhanced response phenotype of eer5-1 was correlated with failure to induce appropriately a subset of ethylene-regulated genes, suggesting that proper ethylene-responsive gene expression is necessary for resetting the ethylene response pathway. eer5-1 represents a mutation that causes an amino acid substitution in a previously uncharacterized gene, which encodes a protein with a PAM [proteasome COP9 initiation factor (PCI/PINT)-associated module] domain similar to those found in components of the COP9 signalosome (CSN). Genetic analysis shows that manifestation of the eer5 mutant phenotype is solely dependent on ethylene signaling, as the ein2-5 eer5-1 double mutant was indistinguishable from ein2-5 in the presence of saturating ethylene concentrations. In contrast, the ein3-1 eer5-1 double mutant displayed characteristics of an enhanced ethylene response, and this suggests that EER5 regulates ethylene signaling independently of EIN3. Analysis of the EER5 protein indicates that it interacts with the C-terminus of EIN2 and with the CSN, suggesting that EER5 serves as a bridge between EIN2 and the modification or degradation of target proteins, including a proposed group of transcriptional repressors, as part of a resetting mechanism during or following ethylene signaling.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Etilenos/metabolismo , Transducción de Señal , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genotipo , Hipocótilo/efectos de los fármacos , Hipocótilo/genética , Mutación , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismoRESUMEN
Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Fosfatidicos/metabolismo , Proteínas Quinasas/metabolismo , Arabidopsis/genética , Ácidos Fosfatidicos/química , Unión Proteica , Proteínas Quinasas/química , Estructura Terciaria de ProteínaRESUMEN
Ethylene is the simplest unsaturated hydrocarbon, yet it has profound effects on plant growth and development, including many agriculturally important phenomena. Analysis of the mechanisms underlying ethylene biosynthesis and signalling have resulted in the elucidation of multistep mechanisms which at first glance appear simple, but in fact represent several levels of control to tightly regulate the level of production and response. Ethylene biosynthesis represents a two-step process that is regulated at both the transcriptional and post-translational levels, thus enabling plants to control the amount of ethylene produced with regard to promotion of responses such as climacteric flower senescence and fruit ripening. Ethylene production subsequently results in activation of the ethylene response, as ethylene accumulation will trigger the ethylene signalling pathway to activate ethylene-dependent transcription for promotion of the response and for resetting the pathway. A more detailed knowledge of the mechanisms underlying biosynthesis and the ethylene response will ultimately enable new approaches to be developed for control of the initiation and progression of ethylene-dependent developmental processes, many of which are of horticultural significance.
Asunto(s)
Etilenos/biosíntesis , Plantas/metabolismo , Desarrollo de la Planta , Transducción de SeñalRESUMEN
A previously-identified Arabidopsis mutant with hypersensitivity to aluminum, als7-1 was studied further to determine the nature of the mutation and subsequently establish the biochemical basis of the increase in Al sensitivity. Physiological analysis revealed that the Al hypersensitivity phenotype is correlated with increased Al uptake and Al-dependent gene expression, indicating that als7-1 has a defect in an Al-exclusion mechanism. Cloning of the als7-1 mutation showed that it negatively affects the gene encoding the putative nucleolar localised ribosomal biogenesis factor SLOW WALKER2, which is required for normal gametogenesis and mitotic progression. Molecular analysis indicated that Al hypersensitivity in als7-1 is correlated with loss of expression of a factor required for S-adenosylmethionine recycling and reduced levels of endogenous polyamines in the mutant. Further analysis shows that Al-dependent root growth inhibition is reversed by addition of exogenous spermine, which is correlated with a significant reduction in Al uptake by spermine treated roots. Endogenous spermine likely functions to compete with Al3+ for binding to extra- and intracellular anionic sites, which suggests that increased spermine levels may be an effective means to improve root growth in Al toxic acid soil environments.
RESUMEN
Ethylene signaling is a complex pathway that has been intensively analyzed partly due to its importance to the manifestation of horticultural phenomena, including fruit ripening and tissue senescence. In order to further our understanding of how this pathway is regulated, a screen for Arabidopsis mutants with increased ethylene response was conducted. From this, a mutant was identified as having a dark-grown hypocotyl that is indistinguishable from Col-0 wt in the presence of the ethylene perception inhibitor AgNO3, yet has extreme responsiveness to even low levels of ethylene. Map-based cloning of the mutation revealed a T-DNA insertion in the coding sequence of the receptor-like kinase FERONIA, which is required for normal pollen tube reception and cell elongation in a currently unknown capacity. In contrast to a previous report, analysis of our feronia knockout mutant shows it also has altered responsiveness to brassinosteroids, with etiolated fer-2 seedlings being partially brassinosteroid insensitive with regard to promotion of hypocotyl elongation. Our results indicate that FERONIA-dependent brassinosteroid response serves to antagonize the effect of ethylene on hypocotyl growth of etiolated seedlings, with loss of proper brassinosteroid signaling disrupting this balance and leading to a greater impact of ethylene on hypocotyl shortening.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/efectos de los fármacos , Etilenos/farmacología , Hipocótilo/efectos de los fármacos , Fosfotransferasas/fisiología , Esteroides Heterocíclicos/farmacología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Northern Blotting , Hipocótilo/genética , Fosfotransferasas/genética , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genéticaRESUMEN
Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.1 PA was shown to inhibit (1) CTR1's protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.
RESUMEN
The eer3-1 loss-of-function mutant, which was identified by screening for Arabidopsis mutant seedlings with an enhanced ethylene response, has both increased sensitivity and profound exaggeration of response to ethylene when visually assessed, yet exhibits partial ethylene insensitivity at the molecular level. The eer3-1 mutation represents a conditional allele with an ethylene-dependent phenotype that results from an amino acid substitution in the previously uncharacterized prohibitin, AtPHB3, with complete loss of EER3 function resulting in an extreme constitutive ethylene response in air. Prohibitins in other organisms have diverse roles including transcriptional regulation, with loss of prohibitin function in this capacity associated with tumour formation in mammals. Subcellular localization of AtPHB3 indicates that it is found in several cellular locations, including the nucleus and throughout the cytoplasm. Genetic analysis demonstrates that EER3 functions downstream of EIN2, since an ein2-5;eer3-2 double mutant has the same profound hypocotyl inhibition phenotype seen with the eer3-2 mutant. Based on the presented work, AtPHB3 probably functions as a positive regulator of expression of a subset of ethylene-regulated genes along with a group of genes required to maintain growth in the presence of ethylene.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Represoras/genética , Plantones/genética , Alquenos/farmacología , Alelos , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/fisiología , Proteínas Fluorescentes Verdes/análisis , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Prohibitinas , Proteínas Recombinantes de Fusión/análisis , Proteínas Represoras/química , Proteínas Represoras/fisiología , Plantones/efectos de los fármacos , Plantones/metabolismo , Alineación de Secuencia , Transducción de Señal/efectos de los fármacosRESUMEN
Aluminum toxicity in acid soils severely limits crop productivity through inhibition of root growth and, consequently, shoot development. Several Arabidopsis mutants were previously identified as having roots with Al hypersensitivity, suggesting that these represent deleterious mutations affecting genes required for either Al tolerance or resistance mechanisms. For this report, the als1-1 mutant was chosen for further characterization. The phenotype of als1-1 is most obviously presented in Al challenged roots, as evidenced by exaggerated root growth inhibition in conjunction with increased expression of Al-responsive genes compared to wt. Using a map-based cloning approach, the als1-1 mutation was isolated and found to represent a deleterious amino acid substitution in a previously uncharacterized half type ABC transporter, At5g39040, which is expressed in a non-Al dependent manner in all organs tested. GUS-dependent analyses revealed that ALS1 expression is primarily localized to the root tip and the vasculature throughout the plant. Concomitant with this, an ALS1: GFP fusion accumulates at the vacuolar membrane of root cells, indicating that ALS1 may be important for intracellular movement of some substrate, possibly chelated Al, as part of a mechanism of Al sequestration.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Aluminio/toxicidad , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Alelos , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacosRESUMEN
eer4 was isolated as an Arabidopsis mutant with an extreme response to ethylene in dark-grown seedlings that was also found to have partial ethylene insensitivity at the level of ethylene-dependent gene expression, including ERF1. Subsequent cloning of eer4 revealed an inappropriate stop codon in a previously uncharacterized TFIID-interacting transcription factor homologous to human TAF12 and yeast TAF61. Genetic and pharmacological analysis demonstrated that the eer4 phenotype is strictly ethylene dependent in seedlings, yet a double mutant with the partially ethylene-insensitive Arabidopsis mutant, ein3-1, had restored ethylene responsiveness, indicating that eer4 also regulates a previously unknown resetting or dampening mechanism for the ethylene signalling pathway. Consistent with the absolute requirement of EER4 for ERF1 expression, biochemical analysis showed that EER4 is localized to the nucleus where it probably recruits EIN3 and probably other transcription factors along with components of the TFIID complex for expression of a subset of genes required for either manifestation or subsequent dampening of the response to ethylene.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Etilenos/metabolismo , Factores de Terminación de Péptidos/metabolismo , Factor de Transcripción TFIID/fisiología , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Codón sin Sentido , Oscuridad , Etilenos/farmacología , Proteínas Fluorescentes Verdes/análisis , Modelos Biológicos , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de Proteína , Transducción de Señal/genética , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismoRESUMEN
Aluminium (Al) toxicity is a global agricultural problem that occurs in acid soil environments and severely limits root growth and crop productivity. The isolation and characterization of a gene, ALS3, which is absolutely required by Arabidopsis seedlings for growth in an Al-toxic environment was reported previously. Since the als3-1 loss-of-function mutant has extreme root growth inhibition even in the presence of very low levels of Al, it was an excellent candidate for using a mutagenesis approach to identify suppressor mutations that would increase either Al resistance or tolerance in Arabidopsis roots. EMS-mutagenized als3-1 seedlings were screened for mutants that could sustain root growth in an Al-containing environment that is highly toxic to als3-1 but not Col-0 wt. This approach resulted in identification of 12 strong suppressor mutants that reversed the als3-1 phenotype and grew as well or better than Col-0 wt in the presence of high levels of Al. Subsequent analysis of three representative suppressor mutants revealed that the phenotype of each probably arises from dominant gain-of-function mutations at the same locus. Detailed analysis of one of these, alt1-1 (Al tolerant), suggests that this mutation positively impacts Al resistance in a manner dependent on pH adjustment rather than enhanced Al exclusion. Identification of these suppressor mutations, should not only further elucidate the biochemical and molecular mechanisms underlying Al toxicity and tolerance but also will develop a collection of mutations that may be useful for engineering crop plants that can grow and thrive in Al-toxic environments.