RESUMO
When faced with salinity stress, plants typically exhibit a slowdown in their growth patterns. Boron (B) is an essential micronutrient for plants that are known to play a critical role in controlling cell wall properties. In this study, we used the model plant Arabidopsis thaliana Col-0 and relevant mutants to explore how the difference in B availability may modulate plant responses to salt stress. There was a visible root growth suppression of Col-0 with the increased salt levels in the absence of B while this growth reduction was remarkably alleviated by B supply. Pharmacological experiments revealed that orthovanadate (a known blocker of H+-ATPase) inhibited root growth at no B condition, but had no effect in the presence of 30 µM B. Salinity stress resulted in a massive K+ loss from mature zones of A. thaliana roots; this efflux was attenuated in the presence of B. Supplemental B also increased the magnitude of net H+ pumping by plant roots. Boron availability was also essential for root halotropism. Interestingly, the aha2Δ57 mutant with active H+-ATPase protein exhibited the same halotropism response as Col-0 while the aha2-4 mutant had a stronger halotropism response (larger bending angle) compared with that of Col-0. Overall, the ameliorative effect of B on the A. thaliana growth under salt stress is based on the H+-ATPase stimulation and a subsequent K+ retention, involving auxin- and ROS-pathways.
RESUMO
Soil salinity is a major environmental constraint affecting the sustainability and profitability of agricultural production systems. Salinity stress tolerance has been present in wild crop relatives but then lost, or significantly weakened, during their domestication. Given the genetic and physiological complexity of salinity tolerance traits, agronomical solutions may be a suitable alternative to crop breeding for improved salinity stress tolerance. One of them is optimizing fertilization practices to assist plants in dealing with elevated salt levels in the soil. In this review, we analyse the causal relationship between the availability of boron (an essential metalloid micronutrient) and plant's adaptive responses to salinity stress at the whole-plant, cellular, and molecular levels, and a possibility of using boron for salt stress mitigation. The topics covered include the impact of salinity and the role of boron in cell wall remodelling, plasma membrane integrity, hormonal signalling, and operation of various membrane transporters mediating plant ionic and water homeostasis. Of specific interest is the role of boron in the regulation of H+-ATPase activity whose operation is essential for the control of a broad range of voltage-gated ion channels. The complex relationship between boron availability and expression patterns and the operation of aquaporins is also discussed.
Assuntos
Boro , Salinidade , Solo , Boro/metabolismo , Solo/química , Adaptação Fisiológica/genética , Tolerância ao Sal/genética , Plantas/metabolismo , Plantas/genética , Regulação da Expressão Gênica de PlantasRESUMO
The increasing prevalence of herbicide-resistant weeds has led to a search for new herbicides that target plant growth processes differing from those targeted by current herbicides. In recent years, some studies have explored the use of natural compounds from microorganisms as potential new herbicides. We previously demonstrated that tenuazonic acid (TeA) from the phytopathogenic fungus Stemphylium loti inhibits the plant plasma membrane (PM) H+-ATPase, representing a new target for herbicides. In this study, we further investigated the mechanism by which TeA inhibits PM H+-ATPase and the effect of the toxin on plant growth using Arabidopsis thaliana. We also studied the biochemical effects of TeA on the PM H+-ATPases from spinach (Spinacia oleracea) and A. thaliana (AHA2) by examining PM H+-ATPase activity under different conditions and in different mutants. Treatment with 200 µM TeA-induced cell necrosis in larger plants and treatment with 10 µM TeA almost completely inhibited cell elongation and root growth in seedlings. We show that the isoleucine backbone of TeA is essential for inhibiting the ATPase activity of the PM H+-ATPase. Additionally, this inhibition depends on the C-terminal domain of AHA2, and TeA binding to PM H+-ATPase requires the Regulatory Region I of the C-terminal domain in AHA2. TeA likely has a higher binding affinity toward PM H+-ATPase than the phytotoxin fusicoccin. Finally, our findings show that TeA retains the H+-ATPase in an inhibited state, suggesting that it could act as a lead compound for creating new herbicides targeting the PM H+-ATPase.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Herbicidas , ATPases Translocadoras de Prótons , Spinacia oleracea , Ácido Tenuazônico , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/enzimologia , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ácido Tenuazônico/metabolismo , Ácido Tenuazônico/farmacologia , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Herbicidas/farmacologia , Herbicidas/química , Spinacia oleracea/efeitos dos fármacos , Spinacia oleracea/crescimento & desenvolvimento , Spinacia oleracea/metabolismoRESUMO
Because of their ability to promote growth, act as biopesticides, and improve abiotic stress tolerance, Trichoderma spp. have been used for plant seed coating. However, the mechanism for the promotion of plant growth remains unknown. In this study, we investigate the effect of fungal extracts on the plant plasma membrane (PM) H+-ATPase, which is essential for plant growth and often a target of plant-associated microbes. We show that Trichoderma harzianum extract increases H+-ATPase activity, and by fractionation and high-resolution mass spectrometry (MS), we identify the activating components trichorzin PA (tPA) II and tPA VI that belong to the class of peptaibols. Peptaibols are nonribosomal peptides that can integrate into membranes and form indiscriminate ion channels, which causes pesticidal activity. To further investigate peptaibol-mediated H+-ATPase activation, we compare the effect of tPA II and VI to that of the model peptaibol alamethicin (AlaM). We show that AlaM increases H+-ATPase turnover rates in a concentration-dependent manner, with a peak in activity measured at 31.25 µM, above which activity decreases. Using fluorescent probes and light scattering, we find that the AlaM-mediated increase in activity is not correlated to increased membrane fluidity or vesicle integrity, whereas the activity decrease at high AlaM concentrations is likely due to PM overloading of AlaM pores. Overall, our results suggest that the symbiosis of fungi and plants, specifically related to peptaibols, is a concentration-dependent balance, where peptaibols do not act only as biocontrol agents but also as plant growth stimulants.
RESUMO
Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its well-known role in tissue maintenance, TIMP-1 has been associated with multiple MMP-independent cytokine-like functions. The protein structure of TIMP-1, with two distinct domains, one interacting with MMPs and another able to bind multiple partners, provides a rationale for this multifunctionality. The identification of CD63 as a cell surface receptor for TIMP-1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMP-1 in cellular signaling. However, several lines of evidence suggest that TIMP-1 may be able to associate with many interaction partners, thus attaining multiple functions. To enable the identification of previously unknown interaction partners that may underpin the core cellular functions of TIMP-1, known as well as unknown, we performed a yeast two-hybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class II-associated invariant chain γ (CD74). We verified that CD74 interacts with TIMP-1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMP-1 and mediates intracellular signaling through the Akt signaling axis in breast cancer cells. These data provide new insights into the complex nature of the functions of TIMP-1 and their potential mechanistic basis.
Assuntos
Neoplasias da Mama , Inibidor Tecidual de Metaloproteinase-1 , Humanos , Feminino , Inibidor Tecidual de Metaloproteinase-1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Metaloproteinases da Matriz/metabolismo , Ligação ProteicaRESUMO
Plasma membrane (PM) H+-ATPases are the electrogenic proton pumps that export H+ from plant and fungal cells to acidify the surroundings and generate a membrane potential. Plant PM H+-ATPases are equipped with a Cterminal autoinhibitory regulatory (R) domain of about 100 amino acid residues, which could not be identified in the PM H+-ATPases of green algae but appeared fully developed in immediate streptophyte algal predecessors of land plants. To explore the physiological significance of this domain, we created in vivo C-terminal truncations of autoinhibited PM H+ATPase2 (AHA2), one of the two major isoforms in the land plant Arabidopsis thaliana. As more residues were deleted, the mutant plants became progressively more efficient in proton extrusion, concomitant with increased expansion growth and nutrient uptake. However, as the hyperactivated AHA2 also contributed to stomatal pore opening, which provides an exit pathway for water and an entrance pathway for pests, the mutant plants were more susceptible to biotic and abiotic stresses, pathogen invasion and water loss, respectively. Taken together, our results demonstrate that pump regulation through the R domain is crucial for land plant fitness and by controlling growth and nutrient uptake might have been necessary already for the successful water-to-land transition of plants.
Assuntos
Arabidopsis , Bombas de Próton , Bombas de Próton/genética , Transporte Biológico , Membrana Celular , Prótons , Água , Arabidopsis/genética , Adenosina TrifosfatasesRESUMO
Small post-translationally modified peptides are gaining increasing attention as important signaling molecules in plant development. In the family of plant peptides containing tyrosine sulfation (PSYs), only PSY1 has been characterized at the mature level as an 18-amino-acid peptide, carrying one sulfated tyrosine, and involved in cell elongation. This review presents seven additional homologs in Arabidopsis all sharing high conservation in the active peptide domain, and it shows that PSY peptides are found in all higher plants and mosses. It is proposed that all eight PSY homologs are post-translationally modified to carry a sulfated tyrosine and that subtilisin-like subtilases (SBTs) are involved in the processing of PSY propeptides. The PSY peptides show differential expression patterns indicating that they serve several distinct functions in plant development. PSY peptides seem to be at least partly regulated at the transcriptional level, as their expression is greatly influenced by developmental factors. Finally, a model including a receptor in addition to PSY1R is proposed.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Desenvolvimento Vegetal/genética , Processamento de Proteína Pós-Traducional/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Peptídeos/genética , Peptídeos/metabolismo , Transdução de Sinais/genéticaRESUMO
We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents.
Assuntos
Antifúngicos/farmacologia , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , ATPases do Tipo-P/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antifúngicos/química , Candida/efeitos dos fármacos , Carbazóis/química , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Células Hep G2 , Humanos , Hidrólise , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , ATPases do Tipo-P/química , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
The plasma membrane H+-ATPase (Pma1) is an essential fungal protein and a proposed target for new antifungal medications. The compounds in a small-molecule library containing â¼191,000 commercially available compounds were screened for their ability to inhibit Saccharomyces cerevisiae plasma membranes containing Pma1. The overall hit rate was 0.2%, corresponding to 407 compounds. These hit compounds were further evaluated for ATPase selectivity and broad-spectrum antifungal activity. Following this work, one Pma1 inhibitor series based on compound 14 and analogs was selected for further evaluation. This compound series was able to depolarize the membrane and inhibit extracellular acidification in intact fungal cells concomitantly with a significant increase in intracellular ATP levels. Collectively, we suggest that these effects may be a common feature of Pma1 inhibitors. Additionally, the work uncovered a dual mechanism for the previously identified cationic peptide BM2, revealing fungal membrane disruption, in addition to Pma1 inhibition. The methods presented here provide a solid platform for the evaluation of Pma1-specific inhibitors in a drug development setting. The present inhibitors could serve as a starting point for the development of new antifungal agents with a novel mode of action.
Assuntos
Antifúngicos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Antifúngicos/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Hep G2 , Humanos , Potenciais da Membrana/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Taking advantage of microwave-assisted synthesis, efficient and expedite procedures for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum sensing and one compound inhibited activity of the las quorum sensing system. To our delight, none of the compounds showed growth inhibitory effects in the tested concentration ranges.
Assuntos
Desenho de Fármacos , Ácido Fusárico/química , Ácido Fusárico/farmacologia , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Modelos Moleculares , Conformação Molecular , ATPases Translocadoras de Prótons/antagonistas & inibidoresRESUMO
While the importance of cell type specificity in plant adaptive responses is widely accepted, only a limited number of studies have addressed this issue at the functional level. We have combined electrophysiological, imaging, and biochemical techniques to reveal the physiological mechanisms conferring higher sensitivity of apical root cells to salinity in barley (Hordeum vulgare). We show that salinity application to the root apex arrests root growth in a highly tissue- and treatment-specific manner. Although salinity-induced transient net Na+ uptake was about 4-fold higher in the root apex compared with the mature zone, mature root cells accumulated more cytosolic and vacuolar Na+, suggesting that the higher sensitivity of apical cells to salt is not related to either enhanced Na+ exclusion or sequestration inside the root. Rather, the above differential sensitivity between the two zones originates from a 10-fold difference in K+ efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Major factors contributing to this poor K+ retention ability are (1) an intrinsically lower H+-ATPase activity in the root apex, (2) greater salt-induced membrane depolarization, and (3) a higher reactive oxygen species production under NaCl and a larger density of reactive oxygen species-activated cation currents in the apex. Salinity treatment increased (2- to 5-fold) the content of 10 (out of 25 detected) amino acids in the root apex but not in the mature zone and changed the organic acid and sugar contents. The causal link between the observed changes in the root metabolic profile and the regulation of transporter activity is discussed.
Assuntos
Aclimatação , Hordeum/enzimologia , Hordeum/fisiologia , Raízes de Plantas/enzimologia , Potássio/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Salinidade , Estresse Fisiológico , Aclimatação/efeitos dos fármacos , Alantoína/farmacologia , Cátions/metabolismo , Hordeum/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Metabolômica , Modelos Biológicos , Especificidade de Órgãos/efeitos dos fármacos , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Curcumina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Saccharomyces cerevisiae/enzimologia , Spinacia oleracea/enzimologia , Curcumina/farmacologia , DiarileptanoidesRESUMO
BACKGROUND: The plant nuclear pore complex has strongly attracted the attention of the scientific community during the past few years, in particular because of its involvement in hormonal and pathogen/symbiotic signalling. In Arabidopsis thaliana, more than 30 nucleoporins have been identified, but only a few of them have been characterized. Among these, AtNUP160, AtNUP96, AtNUP58, and AtTPR have been reported to modulate auxin signalling, since corresponding mutants are suppressors of the auxin resistance conferred by the axr1 (auxin-resistant) mutation. The present work is focused on AtNUP62, which is essential for embryo and plant development. This protein is one of the three nucleoporins (with AtNUP54 and AtNUP58) of the central channel of the nuclear pore complex. RESULTS: AtNUP62 promoter activity was detected in many organs, and particularly in the embryo sac, young germinating seedlings and at the adult stage in stipules of cauline leaves. The atnup62-1 mutant, harbouring a T-DNA insertion in intron 5, was identified as a knock-down mutant. It displayed developmental phenotypes that suggested defects in auxin transport or responsiveness. Atnup62 mutant plantlets were found to be hypersensitive to auxin, at the cotyledon and root levels. The phenotype of the AtNUP62-GFP overexpressing line further supported the existence of a link between AtNUP62 and auxin signalling. Furthermore, the atnup62 mutation led to an increase in the activity of the DR5 auxin-responsive promoter, and suppressed the auxin-resistant root growth and leaf serration phenotypes of the axr1 mutant. CONCLUSION: AtNUP62 appears to be a major negative regulator of auxin signalling. Auxin hypersensitivity of the atnup62 mutant, reminding that of atnup58 (and not observed with other nucleoporin mutants), is in agreement with the reported interaction between AtNUP62 and AtNUP58 proteins, and suggests closely related functions. The effect of AtNUP62 on auxin signalling likely occurs in relation to scaffold proteins of the nuclear pore complex (AtNUP160, AtNUP96 and AtTPR).
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Glicoproteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Mutagênese Insercional , Regiões Promotoras Genéticas , Transdução de Sinais , Transformação GenéticaRESUMO
The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase.
Assuntos
Membrana Celular/metabolismo , Células Vegetais/metabolismo , Fenômenos Fisiológicos Vegetais , ATPases Translocadoras de Prótons/metabolismo , Transporte Biológico , Células Vegetais/microbiologia , Processamento de Proteína Pós-TraducionalRESUMO
Abiotic stresses such as salinity, drought, and flooding severely limit food and fibre production and result in penalties of in excess of US$100 billion per annum to the agricultural sector. Improved abiotic stress tolerance to these environmental constraints via traditional or molecular breeding practices requires a good understanding of the physiological and molecular mechanisms behind roots sensing of hostile soils, as well as downstream signalling cascades to effectors mediating plant adaptive responses to the environment. In this review, we discuss some common mechanisms conferring plant tolerance to these three major abiotic stresses. Central to our discussion are: (i) the essentiality of membrane potential maintenance and ATP production/availability and its use for metabolic versus adaptive responses; (ii) reactive oxygen species and Ca(2+) 'signatures' mediating stress signalling; and (iii) cytosolic K(+) as the common denominator of plant adaptive responses. We discuss in detail how key plasma membrane and tonoplast transporters are regulated by various signalling molecules and processes observed in plants under stress conditions (e.g. changes in membrane potential; cytosolic pH and Ca(2+); reactive oxygen species; polyamines; abscisic acid) and how these stress-induced changes are related to expression and activity of specific ion transporters. The reported results are then discussed in the context of strategies for breeding crops with improved abiotic stress tolerance. We also discuss a classical trade-off between tolerance and yield, and possible avenues for resolving this dilemma.
Assuntos
Adaptação Fisiológica , Proteínas de Membrana Transportadoras/genética , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/genética , Secas , Inundações , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , SalinidadeRESUMO
The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes.
Assuntos
Trifosfato de Adenosina/química , Bicamadas Lipídicas/química , ATPases Translocadoras de Prótons/biossíntese , Trifosfato de Adenosina/biossíntese , Aminoacridinas/química , Arabidopsis/enzimologia , Transporte Biológico Ativo , Regulação Enzimológica da Expressão Gênica , Hidrólise , Isoxazóis/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/isolamento & purificação , Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic P-type plasma membrane H(+)-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H(+)-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H(+)-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Lisofosfolipídeos/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática , Fosforilação , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In this study the role of the plasma membrane (PM) H(+) -ATPase for growth and development of roots as response to nitrogen starvation is studied. It is known that root development differs dependent on the availability of different mineral nutrients. It includes processes such as initiation of lateral root primordia, root elongation and increase of the root biomass. However, the signal transduction mechanisms, which enable roots to sense changes in different mineral environments and match their growth and development patterns to actual conditions in the soil, are still unknown. Most recent comments have focused on one of the essential macroelements, namely nitrogen, and its role in the modification of the root architecture of Arabidopsis thaliana. As yet, not all elements of the signal transduction pathway leading to the perception of the nitrate stimulus, and hence to anatomical changes of the root, which allow for adaptation to variable ion concentrations in the soil, are known. Our data demonstrate that primary and lateral root length were shorter and lower in aha2 mutant lines compared with wild-type plants in response to a variable nitrogen source. This suggests that the PM proton pump AHA2 (Arabidopsis plasma membrane H(+) -ATPase isoform 2) is important for root growth and development during different nitrogen regimes. This is possible by controlling the pH homeostasis in the root during growth and development as shown by pH biosensors.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Nitrogênio/metabolismo , ATPases Translocadoras de Prótons/genética , Adaptação Fisiológica , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/enzimologia , Transporte de Íons , Nitratos/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais/genéticaRESUMO
Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Membrana Celular/enzimologia , Bicamadas Lipídicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Reagentes de Ligações Cruzadas , Ativação Enzimática , Escherichia coli/metabolismo , Isoenzimas/metabolismo , Microscopia Eletrônica de Transmissão , Saccharomyces cerevisiae/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods.