RESUMEN
Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis-acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Hidrolasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Genes Reporteros , Poliamino OxidasaRESUMEN
The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.
Asunto(s)
Retroalimentación Fisiológica , NADPH Oxidasas/metabolismo , Nicotiana/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Estallido Respiratorio , Salinidad , Apoptosis/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estomas de Plantas/citología , Estomas de Plantas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estallido Respiratorio/efectos de los fármacos , Cloruro de Sodio/farmacología , Espermidina/metabolismo , Superóxidos/metabolismo , Nicotiana/efectos de los fármacos , Poliamino OxidasaRESUMEN
Polyamines (PAs) have been considered as important molecules for survival. However, evidence reinforces that PAs are also implicated, directly or indirectly, in pathways regulating programmed cell death (PCD). Direct correlation of PAs with cell death refers to their association with particular biological processes, and their physical contact with molecules or structures involved in cell death. Indirectly, PAs regulate PCD through their metabolic derivatives, such as catabolic and interconversion products. Cytotoxic products of PA metabolism are involved in PCD cascades, whereas it remains largely elusive how PAs directly control pathways leading to PCD. In this review, we present and compare advances in PA-dependent PCD in animals and plants.
Asunto(s)
Apoptosis , Fenómenos Fisiológicos de las Plantas , Poliaminas/metabolismo , Animales , Necrosis , Vacuolas/metabolismoRESUMEN
Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G(1) and G(2) phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N(1)-dimethylthiourea, a hydrogen peroxide (H(2)O(2)) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H(2)O(2) production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H(2)O(2) derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.
Asunto(s)
Poliaminas Biogénicas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Xilema/crecimiento & desarrollo , Fragmentación del ADN , Fluorescencia , Peróxido de Hidrógeno/metabolismo , Microscopía Electrónica de Transmisión , Plantas Modificadas Genéticamente , Nicotiana/crecimiento & desarrollo , Zea mays/crecimiento & desarrolloRESUMEN
Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis thaliana, five PAOs (AtPAO1-5) are present with cytosolic or peroxisomal localization. Here, we present a detailed study of the expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 during seedling and flower growth and development through analysis of promoter activity in AtPAO::ß-glucuronidase (GUS) transgenic Arabidopsis plants. The results reveal distinct expression patterns for each studied member of the AtPAO gene family. AtPAO1 is mostly expressed in the transition region between the meristematic and the elongation zone of roots and anther tapetum, AtPAO2 in the quiescent center, columella initials and pollen, AtPAO3 in columella, guard cells and pollen, and AtPAO5 in the vascular system of roots and hypocotyls. Furthermore, treatment with the plant hormone abscisic acid induced expression of AtPAO1 in root tip and AtPAO2 in guard cells. These data suggest distinct physiological role(s) for each member of the AtPAO gene family.
Asunto(s)
Arabidopsis/enzimología , Flores , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , Cartilla de ADN , Glucuronidasa/genética , Plantas Modificadas Genéticamente , Poliamino OxidasaRESUMEN
Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca(2+) concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca(2+) current: the addition of Spd cannot induce the channel open probability increase in excised outside-out patches, indicating that the effect of Spd in the induction of Ca(2+) currents is exerted via a second messenger. This messenger is hydrogen peroxide (H2O2), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization-activated Ca(2+) -permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca(2+) -permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca(2+) -permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca(2+) influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd-derived H2O2 signals Ca(2+) influx, thereby regulating pollen tube growth.
Asunto(s)
Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Tubo Polínico/metabolismo , Polen/metabolismo , Canales de Potasio/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Microscopía Fluorescente , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Técnicas de Placa-Clamp , Tubo Polínico/crecimiento & desarrollo , Espermidina/metabolismo , Poliamino OxidasaRESUMEN
Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Familia de Multigenes , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Alineación de Secuencia , Especificidad por Sustrato , Poliamino OxidasaRESUMEN
Recent results have identified mitochondria as centers of stress-induced generation of reactive oxygen species in plants. Depolarization of plant mitochondrial membrane during stress results the release of programmed cell death (PCD)-inducing factors in the cytosol in a fashion similar to the onset of animal-like PCD. Herein, we report significant similarities of animal-like PCD and salinity stress-induced plant PCD. Short-term salinity stress (3 h) led to depolarization of the mitochondrial membrane, release of cytochrome c (CYT-c), which was visualized using a contemporary molecular technique, activation of caspase-3 type proteases and the onset of PCD in wild type tobacco plants, Nicotiana tabacum cv. Petit Havana. However, PCD was not manifested during long-term salinity stress (24 h). Interestingly long-term salinity stress led to necrotic-like features, which were accompanied by collapse of respiration, reduction of key components of the respiratory chain, such as CYT-c and alternative oxidase, ATP depletion and high proteolytic activity. The results suggest that salinity stress of tobacco plants in planta leads to the onset of animal-like PCD only during the early stages post-stress, while long-term stress leads to necrotic-like features.
Asunto(s)
Apoptosis , Nicotiana/citología , Salinidad , Estrés Fisiológico , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Citocromos c/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Superóxidos/metabolismo , Factores de Tiempo , Nicotiana/efectos de los fármacos , Nicotiana/enzimologíaRESUMEN
Understanding the molecular mode(s) of plant tolerance to heat stress (HS) is crucial since HS is a potential threat to sustainable agriculture and global crop production. Polyamines (PAs) seem to exert multifaceted effects in plant growth and development and responses to abiotic and biotic stresses, presumably via their homeostasis, chemical interactions and contribution to hydrogen peroxide (H2O2) cellular 'signatures'. Downregulation of the apoplastic POLYAMINE OXIDASE (PAO) gene improved thermotolerance in tobacco (Nicotiana tabacum L.) transgenics. However, in the present work we show that transgenic tobacco plants with antisense-mediated S-ADENOSYL-L-METHIONINE DECARBOXYLASE silencing (AS-NtSAMDC) exhibited enhanced sensitivity and delayed responses to HS which was accompanied by profound injury upon HS removal (recovery), as assessed by phenological, physiological and biochemical characteristics. In particular, the AS-NtSAMDC transgenics exhibited significantly reduced rate of photosynthesis, as well as enzymatic and non-enzymatic antioxidants. These transgenics suffered irreversible damage, which significantly reduced their growth potential upon return to normal conditions. These data reinforce the contribution of increased PA homeostasis to tolerance, and can move forward our understanding on the PA-mediated mechanism(s) conferring tolerance to HS that might be targeted via traditional or biotechnological breeding for developing HS tolerant plants.
Asunto(s)
Peróxido de Hidrógeno , Nicotiana , Carboxiliasas , Regulación de la Expresión Génica de las Plantas , Respuesta al Choque Térmico , Peróxido de Hidrógeno/metabolismo , Fitomejoramiento , Nicotiana/metabolismoRESUMEN
In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.
Asunto(s)
Glutamato Deshidrogenasa/metabolismo , Nicotiana/enzimología , Proteínas de Plantas/metabolismo , Glutamato Deshidrogenasa/genética , Ácido Glutámico/biosíntesis , Espectroscopía de Resonancia Magnética , Nitrógeno/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Compuestos de Amonio Cuaternario/metabolismo , Nicotiana/genéticaRESUMEN
The chaperones, heat shock proteins (HSPs), stabilize proteins to minimize proteotoxic stress, especially during heat stress (HS) and polyamine (PA) oxidases (PAOs) participate in the modulation of the cellular homeostasis of PAs and reactive oxygen species (ROS). An interesting interaction of HSP90s and PAOs was revealed in Arabidopsis thaliana by using the pLFY:HSP90RNAi line against the four AtHSP90 genes encoding cytosolic proteins, the T-DNA Athsp90-1 and Athsp90-4 insertional mutants, the Atpao3 mutant and pharmacological inhibitors of HSP90s and PAOs. Silencing of all cytosolic HSP90 genes resulted in several-fold higher levels of soluble spermidine (S-Spd), acetylated Spd (N8-acetyl-Spd) and acetylated spermine (N1-acetyl-Spm) in the transgenic Arabidopsis thaliana leaves. Heat shock induced increase of soluble-PAs (S-PAs) and soluble hydrolyzed-PAs (SH-PAs), especially of SH-Spm, and more importantly of acetylated Spd and Spm. The silencing of HSP90 genes or pharmacological inhibition of the HSP90 proteins by the specific inhibitor radicicol, under HS stimulatory conditions, resulted in a further increase of PA titers, N8-acetyl-Spd and N1-acetyl-Spm, and also stimulated the expression of PAO genes. The increased PA titers and PAO enzymatic activity resulted in a profound increase of PAO-derived hydrogen peroxide (H2O2) levels, which was terminated by the addition of the PAO-specific inhibitor guazatine. Interestingly, the loss-of-function Atpao3 mutant exhibited increased mRNA levels of selected AtHSP90 genes. Taken together, the results herein reveal a novel function of HSP90 and suggest that HSP90s and PAOs cross-talk to orchestrate PA acetylation, oxidation, and PA/H2O2 homeostasis.
RESUMEN
The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.
Asunto(s)
Nicotiana/enzimología , Nicotiana/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Estallido Respiratorio , Zea mays/enzimología , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Espacio Extracelular/enzimología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Peróxido de Hidrógeno/farmacología , Hidrólisis/efectos de los fármacos , Focalización Isoeléctrica , Isoenzimas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Poliaminas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Superóxidos/metabolismo , Nicotiana/citología , Nicotiana/efectos de los fármacos , Transformación Genética , Transgenes , Vitamina K 3/farmacología , Zea mays/efectos de los fármacos , Poliamino OxidasaRESUMEN
The intra-/intercellular homeostasis of reactive oxygen species (ROS), and especially of superoxides (O2.-) and hydrogen peroxide (O2.-) participate in signalling cascades which dictate developmental processes and reactions to biotic/abiotic stresses. Polyamine oxidases terminally oxidize/back convert polyamines generating H2O2. Recently, an NADPH-oxidase/Polyamine oxidase feedback loop was identified to control oxidative burst under salinity. Thus, the real-time localization/monitoring of ROS in specific cells, such as the guard cells, can be of great interest. Here we present a detailed description of the real-time in vivo monitoring of ROS in the guard cells using H2O2- and O2.- specific fluorescing probes, which can be used for studying ROS accumulation generated from any source, including the amine oxidases-dependent pathway, during development and stress.
Asunto(s)
Especies Reactivas de Oxígeno/metabolismo , Transporte Biológico , Espacio Extracelular/metabolismo , Peróxido de Hidrógeno/metabolismo , Espacio Intracelular/metabolismo , Microscopía Confocal , Oxidación-Reducción , Células Vegetales , Nicotiana/citología , Nicotiana/metabolismoRESUMEN
Polyamines (PAs) and hydrogen peroxide (H2O2), the product of PA oxidation by polyamine oxidase (PAO), are potential players affecting plant growth, development and responses to abiotic/biotic stresses. Genetically modified Nicotiana tabacum plants with altered PA/H2O2 homeostasis due to over/underexpression of the ZmPAO gene (S-ZmPAO/AS-ZmPAO, respectively) were assessed under heat stress (HS). Underexpression of ZmPAO correlates with increased thermotolerance of the photosynthetic machinery and improved biomass accumulation, accompanied by enhanced levels of the enzymatic and non-enzymatic antioxidants, whereas ZmPAO overexpressors exhibit significant impairment of thermotolerance. These data provide important clues on PA catabolism/H2O2/thermotolerance, which merit further exploitation.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nicotiana/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Termotolerancia , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Poliaminas/metabolismo , Zea mays/genética , Poliamino OxidasaRESUMEN
Polyamine (PA) homeostasis is associated with plant development, growth and responses to biotic/abiotic stresses. Apoplastic PA oxidase (PAO) catalyzes the oxidation of PAs contributing to cellular homeostasis of reactive oxygen species (ROS) and PAs. In tobacco, PAs decrease with plant age, while apoplastic PAO activity increases. Our previous results with young transgenic tobacco plants with enhanced/reduced apoplastic PAO activity (S-ZmPAO/AS-ZmPAO, respectively) established the importance of apoplastic PAO in controlling tolerance to short-term salt stress. However, it remains unclear if the apoplastic PAO pathway is important for salt tolerance at later stages of plant development. In this work, we examined whether apoplastic PAO controls also plant development and tolerance of adult plants during long-term salt stress. The AS-ZmPAO plants contained higher Ca2+ during salt stress, showing also reduced chlorophyll content index (CCI), leaf area and biomass but taller phenotype compared to the wild-type plants during salt. On the contrary, the S-ZmPAO had more leaves with slightly greater size compared to the AS-ZmPAO and higher antioxidant genes/enzyme activities. Accumulation of proline in the roots was evident at prolonged stress and correlated negatively with PAO deregulation as did the transcripts of genes mediating ethylene biosynthesis. In contrast to the strong effect of apoplastic PAO to salt tolerance in young plants described previously, the effect it exerts at later stages of development is rather moderate. However, the different phenotypes observed in plants deregulating PAO reinforce the view that apoplastic PAO exerts multifaceted roles on plant growth and stress responses. Our data suggest that deregulation of the apoplastic PAO can be further examined as a potential approach to breed plants with enhanced/reduced tolerance to abiotic stress with minimal associated trade-offs.
Asunto(s)
Nicotiana/crecimiento & desarrollo , Nicotiana/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Cloruro de Sodio/farmacología , Zea mays/enzimología , Ascorbato Peroxidasas/metabolismo , Biomasa , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Catalasa/metabolismo , Electrólitos/metabolismo , Etilenos/biosíntesis , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Homeostasis/efectos de los fármacos , Iones , Fenoles/análisis , Fenotipo , Fotosíntesis/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Prolina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Superóxido Dismutasa/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/genética , Poliamino OxidasaRESUMEN
Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.
RESUMEN
Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions ([Formula: see text] ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and [Formula: see text] . These results suggest that the ratio of [Formula: see text] /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of [Formula: see text] by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.
RESUMEN
Polyamine oxidation is the main catabolic process of polyamines. This process is crucial because not only it participates in the regulation of the endogenous titers of polyamines but also it generates hydrogen peroxide, which can act as a signaling molecule. The recent identification of polyamine oxidases that differ in substrate specificity and mode of action in plants necessitates the use of additional techniques for their characterization based on the determination of the end-product. Herein, we describe the most widely used techniques as well as new techniques that can be used for analysis of the newly identified polyamine oxidases.
Asunto(s)
Pruebas de Enzimas/métodos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Plantas/enzimología , Arabidopsis/enzimología , Fraccionamiento Celular , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Colorimetría , Flavina-Adenina Dinucleótido/metabolismo , Peróxido de Hidrógeno/metabolismo , Cinética , Proteínas de Unión a Maltosa/aislamiento & purificación , Biosíntesis de Proteínas , Putrescina/metabolismo , Pirroles/metabolismo , Radiometría , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espermidina/metabolismo , Especificidad por Sustrato , Poliamino OxidasaRESUMEN
Recently we showed that ABA is at least partly responsible for the induction of the polyamine exodus pathway in Vitis vinifera plants. Both sensitive and tolerant plants employ this pathway to orchestrate stress responses, differing between stress adaptation and programmed cell death. Herein we show that ABA is an upstream signal for the induction of the polyamine catabolic pathway in Vitis vinifera. Thus, amine oxidases are producing H2O2 which signals stomata closure. Moreover, the previously proposed model for the polyamine catabolic pathway is updated and discussed.
Asunto(s)
Ácido Abscísico/metabolismo , Peróxido de Hidrógeno/metabolismo , Monoaminooxidasa/metabolismo , Estomas de Plantas/fisiología , Poliaminas/metabolismo , Estrés Fisiológico , Vitis/fisiología , Adaptación Fisiológica , Muerte Celular , Modelos Biológicos , Transducción de Señal , Vitis/metabolismoRESUMEN
Polyamines (PAs) have been suggested to be implicated in plant responses to abiotic and biotic stress. Grapevine is a model perennial plant species whose cultivars respond differently to osmotic stress. In this study, we used two cultivars, one sensitive (S) and one tolerant (T) to drought. In adult vines subjected to drought under greenhouse conditions, total PAs were significantly lower in the control T- and higher in the control S-genotype and significantly increased or decreased, respectively, post-treatment. Soluble Put and Spd exhibited the greatest increase on d 8 post-treatment in the T- but not in the S-genotype, which accumulated soluble Spm. Abscisic acid (ABA) was differentially accumulated in T- and S-genotypes under drought conditions, and activated the PA biosynthetic pathway, which in turn was correlated with the differential increases in PA titers. In parallel, polyamine oxidases (PAOs) increased primarily in the S-genotype. ABA at least partially induced PA accumulation and exodus into the apoplast, where they were oxidized by the apoplastic amine oxidases (AOs), producing H2O2, which signaled secondary stress responses. The results here show that the ABA signaling pathway integrates PAs and AOs to regulate the generation of H2O2, which signals further stress responses or the PCD syndrome.