Publications by authors named "María C Romero-Puertas"

52 Publications

Targeting redox metabolism of the maize-Azospirillum brasilense interaction exposed to arsenic-affected groundwater.

Physiol Plant 2021 Jul 30. Epub 2021 Jul 30.

Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas (INIAB-CONICET), Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Córdoba, Argentina.

Arsenic in groundwater constitutes an agronomic problem due to its potential accumulation in the food chain. Among the agro-sustainable tools to reduce metal(oid)s toxicity, the use of plant growth-promoting bacteria (PGPB) becomes important. For that, and based on previous results in which significant differences of As translocation were observed when inoculating maize plants with Az39 or CD Azospirillum strains, we decided to decipher the redox metabolism changes and the antioxidant system response of maize plants inoculated when exposed to a realistic arsenate (As ) dose. Results showed that As caused morphological changes in the root exodermis. Photosynthetic pigments decreased only in CD inoculated plants, while oxidative stress evidence was detected throughout the plant, regardless of the assayed strain. The antioxidant response was strain-differential since only CD inoculated plants showed an increase in superoxide dismutase, glutathione S-transferase (GST), and glutathione reductase (GR) activities while other enzymes showed the same behavior irrespective of the inoculated strain. Gene expression assays reported that only GST23 transcript level was upregulated by arsenate, regardless of the inoculated strain. As diminished the glutathione (GSH) content of roots inoculated with the Az39 strain, and CD inoculated plants showed a decrease of oxidized GSH (GSSG) levels. We suggest a model in which the antioxidant response of the maize-diazotrophs system is modulated by the strain and that GSH plays a central role acting mainly as a substrate for GST. These findings generate knowledge for a suitable PGPB selection, and its scaling to an effective bioinoculant formulation for maize crops exposed to adverse environmental conditions.
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http://dx.doi.org/10.1111/ppl.13514DOI Listing
July 2021

Rhizobial Volatiles: Potential New Players in the Complex Interkingdom Signaling With Legumes.

Front Plant Sci 2021 22;12:698912. Epub 2021 Jun 22.

Estación Experimental del Zaidín, CSIC, Granada, Spain.

Bacteria release a wide range of volatile compounds that play important roles in intermicrobial and interkingdom communication. Volatile metabolites emitted by rhizobacteria can promote plant growth and increase plant resistance to both biotic and abiotic stresses. Rhizobia establish beneficial nitrogen-fixing symbiosis with legume plants in a process starting with a chemical dialog in the rhizosphere involving various diffusible compounds. Despite being one of the most studied plant-interacting microorganisms, very little is known about volatile compounds produced by rhizobia and their biological/ecological role. Evidence indicates that plants can perceive and respond to volatiles emitted by rhizobia. In this perspective, we present recent data that open the possibility that rhizobial volatile compounds have a role in symbiotic interactions with legumes and discuss future directions that could shed light onto this area of investigation.
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http://dx.doi.org/10.3389/fpls.2021.698912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258405PMC
June 2021

Nitric oxide signalling in the root is required for MYB72-dependent systemic resistance induced by Trichoderma volatiles in Arabidopsis.

J Exp Bot 2021 Jun 15. Epub 2021 Jun 15.

Plant-Microorganism Interaction Unit, Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC), Cordel de Merinas, 40, 37008, Salamanca, Spain.

Volatile compounds (VCs) of Trichoderma fungi trigger an induced systemic resistance (ISR) in Arabidopsis that is effective against a broad spectrum of pathogens. The root-specific transcription factor MYB72 is an early regulator of ISR and also controls the activation of iron deficiency responses. Nitric oxide (NO) is involved in the regulation of MYB72-dependent iron deficiency responses in Arabidopsis roots, but the role of NO in the regulation of MYB72 and ISR by Trichoderma VCs remains unexplored. Using in vitro bioassays, Trichoderma VCs were applied to Arabidopsis seedlings. Plant perception of Trichoderma VCs triggered a burst of NO in Arabidopsis roots. By suppressing this burst using a NO scavenger, we show the involvement of NO in Trichoderma VCs-mediated regulation of MYB72. Using a NO scavenger and the Arabidopsis lines myb72 and nia1nia2 in in planta bioassays, we demonstrate that NO-signalling is required in the roots for the activation of Trichoderma VCs-mediated ISR against the leaf pathogen Botrytis cinerea. Analysis of the defence-related genes PR1 and PDF1.2 point to the involvement of root NO in priming leaves for enhanced defence. Our results support a key role of root NO-signalling in the regulation of MYB72 during the activation of ISR by Trichoderma VCs.
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http://dx.doi.org/10.1093/jxb/erab294DOI Listing
June 2021

An update on redox signals in plant responses to biotic and abiotic stress crosstalk: insights from cadmium and fungal pathogen interactions.

J Exp Bot 2021 Aug;72(16):5857-5875

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estacion Experimental del Zaidin (EEZ), Consejo Superior de Investigaciones Cientificas (CSIC), Apartado 419, 18080 Granada, Spain.

Complex signalling pathways are involved in plant protection against single and combined stresses. Plants are able to coordinate genome-wide transcriptional reprogramming and display a unique programme of transcriptional responses to a combination of stresses that differs from the response to single stresses. However, a significant overlap between pathways and some defence genes in the form of shared and general stress-responsive genes appears to be commonly involved in responses to multiple biotic and abiotic stresses. Reactive oxygen and nitrogen species, as well as redox signals, are key molecules involved at the crossroads of the perception of different stress factors and the regulation of both specific and general plant responses to biotic and abiotic stresses. In this review, we focus on crosstalk between plant responses to biotic and abiotic stresses, in addition to possible plant protection against pathogens caused by previous abiotic stress. Bioinformatic analyses of transcriptome data from cadmium- and fungal pathogen-treated plants focusing on redox gene ontology categories were carried out to gain a better understanding of common plant responses to abiotic and biotic stresses. The role of reactive oxygen and nitrogen species in the complex network involved in plant responses to changes in their environment is also discussed.
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http://dx.doi.org/10.1093/jxb/erab271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355756PMC
August 2021

Peroxisomes as Redox-Signaling Nodes in Intracellular Communication and Stress Responses.

Plant Physiol 2021 Feb 15. Epub 2021 Feb 15.

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín-CSIC, Profesor Albareda 1, 18008 Granada, Spain.

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http://dx.doi.org/10.1093/plphys/kiab060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154099PMC
February 2021

Role of potassium transporter KUP8 in plant responses to heavy metals.

Physiol Plant 2021 Sep 10;173(1):180-190. Epub 2021 Feb 10.

Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Heavy metal concentrations, which have been increasing over the last 200 years, affect soil quality and crop yields. These elements are difficult to eliminate from soils and may constitute a human health hazard by entering the food chain. Recently, we obtained a selection of mutants with different degrees of tolerance to a mixture of heavy metals (HMmix) in order to gain a deeper insight into the underlying mechanism regulating plant responses to these elements. In this study, we characterized the mutant obtained Atkup8 (in this work, Atkup8-2), which showed one of the most resistant phenotypes, as determined by seedling root length. Atkup8-2 is affected in the potassium transporter KUP8, a member of the high-affinity K uptake family KUP/HAK/KT. Atkup8-2 mutants, which are less affected as measured by seedling root length under HMmix conditions, showed a resistant phenotype with respect to WT seedlings which, despite their delayed growth, are able to develop true leaves at levels similar to those under control conditions. Adult Atkup8-2 plants had a higher fresh weight than WT plants, a resistant phenotype under HMmix stress conditions and lower levels of oxidative damage. KUP8 did not appear to be involved in heavy metal or macro- and micro-nutrient uptake and translocation from roots to leaves, as total concentrations of these elements were similar in both Atkup8-2 and WT plants. However, alterations in cellular K homeostasis in this mutant cannot be ruled out.
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http://dx.doi.org/10.1111/ppl.13345DOI Listing
September 2021

Nitric Oxide, an Essential Intermediate in the Plant-Herbivore Interaction.

Front Plant Sci 2020 8;11:620086. Epub 2021 Jan 8.

Centro de Biotecnologia y Genómica de Plantas, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Universidad Politécnica de Madrid, Madrid, Spain.

Reactive nitrogen species (RNS), mainly nitric oxide (NO), are highly reactive molecules with a prominent role in plant response to numerous stresses including herbivores, although the information is still very limited. This perspective article compiles the current progress in determining the NO function, as either a signal molecule, a metabolic intermediate, or a toxic oxidative product, as well as the contribution of molecules associated with NO metabolic pathway in the generation of plant defenses against phytophagous arthropods, in particular to insects and acari.
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http://dx.doi.org/10.3389/fpls.2020.620086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819962PMC
January 2021

Nitric oxide is essential for cadmium-induced peroxule formation and peroxisome proliferation.

Plant Cell Environ 2020 10 19;43(10):2492-2507. Epub 2020 Aug 19.

Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Granada, Spain.

Nitric oxide (NO) and nitrosylated derivatives are produced in peroxisomes, but the impact of NO metabolism on organelle functions remains largely uncharacterised. Double and triple NO-related mutants expressing cyan florescent protein (CFP)-SKL (nox1 × px-ck and nia1 nia2 × px-ck) were generated to determine whether NO regulates peroxisomal dynamics in response to cadmium (Cd) stress using confocal microscopy. Peroxule production was compromised in the nia1 nia2 mutants, which had lower NO levels than the wild-type plants. These findings show that NO is produced early in the response to Cd stress and was involved in peroxule production. Cd-induced peroxisomal proliferation was analysed using electron microscopy and by the accumulation of the peroxisomal marker PEX14. Peroxisomal proliferation was inhibited in the nia1 nia2 mutants. However, the phenotype was recovered by exogenous NO treatment. The number of peroxisomes and oxidative metabolism were changed in the NO-related mutant cells. Furthermore, the pattern of oxidative modification and S-nitrosylation of the catalase (CAT) protein was changed in the NO-related mutants in both the absence and presence of Cd stress. Peroxisome-dependent signalling was also affected in the NO-related mutants. Taken together, these results show that NO metabolism plays an important role in peroxisome functions and signalling.
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http://dx.doi.org/10.1111/pce.13855DOI Listing
October 2020

Peroxisomal Metabolism and Dynamics at the Crossroads Between Stimulus Perception and Fast Cell Responses to the Environment.

Front Cell Dev Biol 2020 26;8:505. Epub 2020 Jun 26.

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.

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http://dx.doi.org/10.3389/fcell.2020.00505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333514PMC
June 2020

Unraveling the impact of arsenic on the redox response of peanut plants inoculated with two different Bradyrhizobium sp. strains.

Chemosphere 2020 Nov 21;259:127410. Epub 2020 Jun 21.

Instituto de Investigaciones Agrobiotecnológicas - Consejo Nacional de Investigaciones Científicas y Técnicas (INIAB-CONICET), Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto (UNRC), Ruta 36, Km 601, X5800, Río Cuarto, Córdoba, Argentina. Electronic address:

Arsenic (As) can be present naturally in groundwater from peanut fields, constituting a serious problem, as roots can accumulate and mobilize the metalloid to their edible parts. Understanding the redox changes in the legume exposed to As may help to detect potential risks to human health and recognize tolerance mechanisms. Thirty-days old peanut plants inoculated with Bradyrhizobium sp. strains (SEMIA6144 or C-145) were exposed to a realistic arsenate concentration, in order to unravel the redox response and characterize the oxidative stress indexes. Thus, root anatomy, reactive oxygen species detection by fluorescence microscopy and, ROS histochemical staining along with the NADPH oxidase activity were analyzed. Besides, photosynthetic pigments and damage to lipids and proteins were determined as oxidative stress indicators. Results showed that at 3 μM As, the cross-section areas of peanut roots were augmented; NADPH oxidase activity was significantly increased and O˙¯and HO accumulated in leaves and roots. Likewise, an increase in the lipid peroxidation and protein carbonyls was also observed throughout the plant regardless the inoculated strain, while chlorophylls and carotenes were increased only in those inoculated with Bradyrhizobium sp. C-145. Interestingly, the oxidative burst, mainly induced by the NADPH oxidase activity, and the consequent oxidative stress was strain-dependent and organ-differential. Additionally, As modifies the root anatomy, acting as a possibly first defense mechanism against the metalloid entry. All these findings allowed us to conclude that the redox response of peanut is conditioned by the rhizobial strain, which contributes to the importance of effectively formulating bioinoculants for this crop.
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http://dx.doi.org/10.1016/j.chemosphere.2020.127410DOI Listing
November 2020

Low endogenous NO levels in roots and antioxidant systems are determinants for the resistance of Arabidopsis seedlings grown in Cd.

Environ Pollut 2020 Jan 15;256:113411. Epub 2019 Oct 15.

Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín-CSIC, Granada, Spain. Electronic address:

Cadmium (Cd), which is a toxic non-essential heavy metal capable of entering plants and thus the food chain, constitutes a major environmental and health concern worldwide. An understanding of the tools used by plants to overcome Cd stress could lead to the production of food crops with lower Cd uptake capacity and of plants with greater Cd uptake potential for phytoremediation purposes in order to restore soil efficiency in self-sustaining ecosystems. The signalling molecule nitric oxide (NO), whose function remains unclear, has recently been involved in responses to Cd stress. Using different mutants, such as nia1nia2, nox1, argh1-1 and Atnoa1, which were altered in NO metabolism, we analysed various parameters related to reactive oxygen and nitrogen species (ROS/RNS) metabolism and seedling fitness following germination and growth under Cd treatment conditions for seven days. Seedling roots were the most affected, with an increase in ROS and RNS observed in wild type (WT) seedling roots, leading to increased oxidative damage and fitness loss. Mutants that showed lower NO levels in seedling roots under Cd stress were more resistant than WT seedlings due to the maintenance of antioxidant systems which protect against oxidative damage.
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http://dx.doi.org/10.1016/j.envpol.2019.113411DOI Listing
January 2020

Multilevel Regulation of Peroxisomal Proteome by Post-Translational Modifications.

Int J Mol Sci 2019 Oct 1;20(19). Epub 2019 Oct 1.

Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain.

Peroxisomes, which are ubiquitous organelles in all eukaryotes, are highly dynamic organelles that are essential for development and stress responses. Plant peroxisomes are involved in major metabolic pathways, such as fatty acid β-oxidation, photorespiration, ureide and polyamine metabolism, in the biosynthesis of jasmonic, indolacetic, and salicylic acid hormones, as well as in signaling molecules such as reactive oxygen and nitrogen species (ROS/RNS). Peroxisomes are involved in the perception of environmental changes, which is a complex process involving the regulation of gene expression and protein functionality by protein post-translational modifications (PTMs). Although there has been a growing interest in individual PTMs in peroxisomes over the last ten years, their role and cross-talk in the whole peroxisomal proteome remain unclear. This review provides up-to-date information on the function and crosstalk of the main peroxisomal PTMs. Analysis of whole peroxisomal proteomes shows that a very large number of peroxisomal proteins are targeted by multiple PTMs, which affect redox balance, photorespiration, the glyoxylate cycle, and lipid metabolism. This multilevel PTM regulation could boost the plasticity of peroxisomes and their capacity to regulate metabolism in response to environmental changes.
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http://dx.doi.org/10.3390/ijms20194881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801620PMC
October 2019

triggers an early and transient burst of nitric oxide and the upregulation of in tomato roots.

Plant Signal Behav 2019 17;14(9):1640564. Epub 2019 Jul 17.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín - Consejo Superior de Investigaciones Científicas , Granada , Spain.

We recently demonstrated that nitric oxide (NO) accumulation and transcriptional regulation are early components of the regulatory pathway that is activated in tomato roots during the onset of the mycorrhizal symbiosis between and tomato roots. We further showed that the mycorrhizal interaction was associated with a specific NO-related signature, different from that triggered by the pathogen . Here, we extend our investigation by exploring the NO- and related root responses elicited by another root mutualistic endosymbiotic fungus: T-78. By using T-78 -grown cultures, we found that T-78 triggered an early and transient burst of NO in tomato roots during the first hours after the interaction. T-78 also elicited the early upregulation of , which was maintained during the analyzed timespan. By using glass-house bioassays, we found that in a well-established tomato-T-78 symbiosis, NO root levels were maintained at basal level while expression remained upregulated. Our results demonstrate that the T-78 symbiosis is associated with a rapid and transient burst of NO in the host roots and the transcriptional activation of from early stages of the interaction until the establishment of the symbiosis, most likely to control NO levels and favor the mutualistic symbiosis.
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http://dx.doi.org/10.1080/15592324.2019.1640564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768279PMC
June 2020

Nitric oxide in plant-fungal interactions.

J Exp Bot 2019 08;70(17):4489-4503

Plant-Microorganism Interaction Unit, Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC), Salamanca, Spain.

Whilst many interactions with fungi are detrimental for plants, others are beneficial and result in improved growth and stress tolerance. Thus, plants have evolved sophisticated mechanisms to restrict pathogenic interactions while promoting mutualistic relationships. Numerous studies have demonstrated the importance of nitric oxide (NO) in the regulation of plant defence against fungal pathogens. NO triggers a reprograming of defence-related gene expression, the production of secondary metabolites with antimicrobial properties, and the hypersensitive response. More recent studies have shown a regulatory role of NO during the establishment of plant-fungal mutualistic associations from the early stages of the interaction. Indeed, NO has been recently shown to be produced by the plant after the recognition of root fungal symbionts, and to be required for the optimal control of mycorrhizal symbiosis. Although studies dealing with the function of NO in plant-fungal mutualistic associations are still scarce, experimental data indicate that different regulation patterns and functions for NO exist between plant interactions with pathogenic and mutualistic fungi. Here, we review recent progress in determining the functions of NO in plant-fungal interactions, and try to identify common and differential patterns related to pathogenic and mutualistic associations, and their impacts on plant health.
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http://dx.doi.org/10.1093/jxb/erz289DOI Listing
August 2019

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Plant Cell Environ 2019 09 26;42(9):2696-2714. Epub 2019 Jul 26.

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, CSIC, Granada, 18008, Spain.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.
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http://dx.doi.org/10.1111/pce.13597DOI Listing
September 2019

Role of nitric oxide in plant responses to heavy metal stress: exogenous application versus endogenous production.

J Exp Bot 2019 08;70(17):4477-4488

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado, Granada, Spain.

Anthropogenic activities, such as industrial processes, mining, and agriculture, lead to an increase in heavy metal concentrations in soil, water, and air. Given their stability in the environment, heavy metals are difficult to eliminate and can constitute a human health risk by entering the food chain through uptake by crop plants. An excess of heavy metals is toxic for plants, which have various mechanisms to prevent their accumulation. However, once metals enter the plant, oxidative damage sometimes occurs, which can lead to plant death. Initial production of nitric oxide (NO), which may play a role in plant perception, signalling, and stress acclimation, has been shown to protect against heavy metals. Very little is known about NO-dependent mechanisms downstream from signalling pathways in plant responses to heavy metal stress. In this review, using bioinformatic techniques, we analyse studies of the involvement of NO in plant responses to heavy metal stress, its possible role as a cytoprotective molecule, and its relationship with reactive oxygen species. Some conclusions are drawn and future research perspectives are outlined to further elucidate the signalling mechanisms underlying the role of NO in plant responses to heavy metal stress.
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http://dx.doi.org/10.1093/jxb/erz184DOI Listing
August 2019

Nitric oxide and phytoglobin PHYTOGB1 are regulatory elements in the Solanum lycopersicum-Rhizophagus irregularis mycorrhizal symbiosis.

New Phytol 2019 08 5;223(3):1560-1574. Epub 2019 Jul 5.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín - Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, Granada, 18008, Spain.

The regulatory role of nitric oxide (NO) and phytoglobins in plant response to pathogenic and mutualistic microbes has been evidenced. However, little is known about their function in the arbuscular mycorrhizal (AM) symbiosis. We investigated whether NO and phytoglobin PHYTOGB1 are regulatory components in the AM symbiosis. Rhizophagus irregularis in vitro-grown cultures and tomato plants were used to monitor AM-associated NO-related root responses as compared to responses triggered by the pathogen Fusarium oxysporum. A genetic approach was conducted to understand the role of PHYTOGB1 on NO signaling during both interactions. After a common early peak in NO levels in response to both fungi, a specific NO accumulation pattern was triggered in tomato roots during the onset of the AM interaction. PHYTOGB1 was upregulated by the AM interaction. By contrast, the pathogen triggered a continuous NO accumulation and a strong downregulation of PHYTOGB1. Manipulation of PHYTOGB1 levels in overexpressing and silenced roots led to a deregulation of NO levels and altered mycorrhization and pathogen infection. We demonstrate that the onset of the AM symbiosis is associated with a specific NO-related signature in the host root. We propose that NO regulation by PHYTOGB1 is a regulatory component of the AM symbiosis.
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http://dx.doi.org/10.1111/nph.15898DOI Listing
August 2019

Detection of Reactive Oxygen and Nitrogen Species (ROS/RNS) During Hypersensitive Cell Death.

Methods Mol Biol 2018 ;1743:97-105

Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín (CSIC), Granada, Spain.

Reactive oxygen and nitrogen species (ROS/RNS) are signaling molecules involved in a plethora of physiological processes in plants. Especially, ROS and nitric oxide (NO) are key players that are required for programmed cell death (PCD). The PCD associated with the hypersensitive response (HR) has been well characterized and the role of HO and NO as key signaling molecules inducing HR has been established. Localization of ROS and NO production in plant tissues in response to pathogens can be imaged by confocal laser microscopy by using specific fluorescent probes. Deciphering the time and spatial regulation of ROS and NO is very important to establish the cellular response of plants to adverse conditions. This chapter is mainly focused on the imaging of ROS and RNS accumulation in vivo in plant tissues undergoing PCD.
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http://dx.doi.org/10.1007/978-1-4939-7668-3_9DOI Listing
January 2019

Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum.

Redox Biol 2018 04 12;14:409-416. Epub 2017 Oct 12.

Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, 18008 Granada, Spain. Electronic address:

Redox regulation is of great importance in chloroplasts. Many chloroplast enzymes, such as those belonging to the Calvin-Benson cycle (CBC), have conserved regulatory cysteines which form inhibitory disulphide bridges when physiological conditions become unfavourable. Amongst these enzymes, cFBP1, the CBC fructose-1,6-bisphosphatase (FBPase) isoform, is well known to be redox activated by thioredoxin f through the reduction of a disulphide bridge involving Cys153 and Cys173. Moreover, data obtained during recent years point to S-nitrosylation as another redox post-translational modification putatively regulating an increasing number of plant enzymes, including cFBP1. In this study we have shown that the Pisum sativum cFBP1 can be efficiently S-nitrosylated by GSNO and SNAP, triggering the formation of the regulatory disulphide. Using in vivo experiments with P. sativum we have established that cFBP1 S-nitrosylation only occurs during the light period and we have elucidated by activity assays with Cys-to-Ser mutants that this enzyme may be inactivated through the S-nitrosylation of Cys153. Finally, in the light of the new data, we have proposed an extended redox-regulation model by integrating the S-nitrosylation and the TRX f-mediated regulation of cFBP1.
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http://dx.doi.org/10.1016/j.redox.2017.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651545PMC
April 2018

Screening Arabidopsis mutants in genes useful for phytoremediation.

J Hazard Mater 2017 Aug 7;335:143-151. Epub 2017 Apr 7.

Department of Biochemistry, Cell and Molecular Biology of Plants, EEZ, CSIC, Granada, Spain. Electronic address:

Emissions of heavy metals have risen over the past 200 years and significantly exceed those from natural sources. Phytoremediation strategies may be able to recover soil productivity in self-sustaining ecosystems; however, our knowledge of the molecular mechanisms involved in plant heavy-metal perception and signalling is scarce. The aim of this study was to assemble a "molecular tool box" of genes useful for phytoremediation. To identify mutants with different heavy-metal-tolerance, we first selected a medium from mixtures containing three metals based on their presence in two Spanish mining areas and then screened about 7000 lines of Arabidopsis T-DNA mutants and found 74 lines more resistant and 56 more susceptible than the wild type (WT). Classification of the genes showed that they were mainly linked to transport, protein modification and signalling, with RNA metabolism being the most representative category in the resistant phenotypes and protein metabolism in the sensitive ones. We have characterized one resistant mutant, Athpp9 and one sensitive, Atala4. These mutants showed differences in growth and metal translocation. Additionally, we found that these mutants keep their phenotype in amended former soils, suggesting that these genes may be useful for phytoremediation and the recovery of contaminated soils.
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http://dx.doi.org/10.1016/j.jhazmat.2017.04.021DOI Listing
August 2017

Leaf epinasty and auxin: A biochemical and molecular overview.

Plant Sci 2016 Dec 6;253:187-193. Epub 2016 Oct 6.

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008, Granada, Spain.

Leaf epinasty involves the downward bending of leaves as a result of disturbances in their growth, with a greater expansion in adaxial cells as compared to abaxial surface cells. The co-ordinated anisotropy of growth in epidermal, palisade mesophyll and vascular tissues contributes to epinasty. This phenotype, which is regulated by auxin (indole-3-acetic acid, IAA), controls plant cell division and elongation by regulating the expression of a vast number of genes. Other plant hormones, such as ethylene, abscisic acid and brassinosteroids, also regulate epinasty and hyponasty. Reactive oxygen species (ROS) accumulation induced by auxins and 2,4-dichlorophenoxyacetic acid (2,4-D) triggers epinasty. The role of ROS and nitric oxide (NO) in the regulation of epinasty has recently been established. Thus, treatment with synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) induces disturbances in the actin cytoskeleton through ROS and NO-dependent post-translational modifications in actin by carbonylation and S-nitrosylation, which cause a reduction in the actin filament. Reorientation of microtubules has become a major feature of the response to auxin. The cytoskeleton is therefore a key player in epinastic development.
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http://dx.doi.org/10.1016/j.plantsci.2016.10.002DOI Listing
December 2016

Peroxisomes Extend Peroxules in a Fast Response to Stress via a Reactive Oxygen Species-Mediated Induction of the Peroxin PEX11a.

Plant Physiol 2016 07 13;171(3):1665-74. Epub 2016 May 13.

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín-CSIC, Profesor Albareda 1, 18008 Granada, Spain (M.R.-S.; M.C.R.-P.; M.S.-F.; L.M.S.); and Department of Energy Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 (J.H.)

Peroxisomes are highly dynamic and metabolically active organelles that play an important role in cellular functions, including reactive oxygen species (ROS) metabolism. Peroxisomal dynamics, such as the proliferation, movement, and production of dynamic extensions called peroxules, have been associated with ROS in plant cells. However, the function and regulation of peroxules are largely unknown. Using confocal microscopy, we have shown that treatment of Arabidopsis leaves with the heavy metal cadmium produces time course-dependent changes in peroxisomal dynamics, starting with peroxule formation, followed by peroxisome proliferation, and finally returning to the normal morphology and number. These changes during Cd treatment were regulated by NADPH oxidase (C and F)-related ROS production. Peroxule formation is a general response to stimuli such as arsenic and is regulated by peroxin 11a (PEX11a), as Arabidopsis pex11a RNAi lines are unable to produce peroxules under stress conditions. The pex11a line showed higher levels of lipid peroxidation content and lower expression of genes involved in antioxidative defenses and signaling, suggesting that these extensions are involved in regulating ROS accumulation and ROS-dependent gene expression in response to stress. Our results demonstrate that PEX11a and peroxule formation play a key role in regulating stress perception and fast cell responses to environmental cues.
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http://dx.doi.org/10.1104/pp.16.00648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936588PMC
July 2016

Nitric Oxide Level Is Self-Regulating and Also Regulates Its ROS Partners.

Front Plant Sci 2016 17;7:316. Epub 2016 Mar 17.

Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín (Consejo Superior de Investigaciones Científicas) Granada, Spain.

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http://dx.doi.org/10.3389/fpls.2016.00316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795008PMC
March 2016

S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth.

Nat Commun 2015 Oct 23;6:8669. Epub 2015 Oct 23.

Dpto. de Microbiología y Genética, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), Facultad de Biología, Universidad de Salamanca, C/ Río Duero 12, Salamanca 37185, Spain.

Plant survival depends on seed germination and progression through post-germinative developmental checkpoints. These processes are controlled by the stress phytohormone abscisic acid (ABA). ABA regulates the basic leucine zipper transcriptional factor ABI5, a central hub of growth repression, while the reactive nitrogen molecule nitric oxide (NO) counteracts ABA during seed germination. However, the molecular mechanisms by which seeds sense more favourable conditions and start germinating have remained elusive. Here we show that ABI5 promotes growth via NO, and that ABI5 accumulation is altered in genetic backgrounds with impaired NO homeostasis. S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases, and promotes seed germination. Conversely, mutation of ABI5 at cysteine-153 deregulates protein stability and inhibition of seed germination by NO depletion. These findings suggest an inverse molecular link between NO and ABA hormone signalling through distinct posttranslational modifications of ABI5 during early seedling development.
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http://dx.doi.org/10.1038/ncomms9669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4639896PMC
October 2015

Experimental evidences of the NO action on a recombinant PrxII F from pea plant and its effect preventing the citrate synthase aggregation.

Data Brief 2015 Jun 26;3:108-12. Epub 2015 Feb 26.

CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain.

S-nitrosylation is emerging as a key post-translational protein modification for the transduction of NO as a signaling molecule in plants. This data article supports the research article entitled "Functional and structural changes in plant mitochondrial PrxII F caused by NO" [1]. To identify the Cys residues of the recombinant PrxII F modified after the treatment with S-nitrosylating agents we performed the LC ESI-QTOF tandem MS and MALDI peptide mass fingerprinting analysis. Change in A 650 nm was monitored to estimate the thermal aggregation of citrate synthase in the presence S-nitrosylated PrxII F. The effect of the temperature on the oligomerization pattern and aggregation of PrxII F was analysed by SDS-PAGE and changes in absorbance at 650 nm, respectively.
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http://dx.doi.org/10.1016/j.dib.2015.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510073PMC
June 2015

The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility.

J Exp Bot 2015 May 7;66(10):2869-76. Epub 2015 Mar 7.

Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, E-18008 Granada, Spain.

Successful sexual reproduction often relies on the ability of plants to recognize self- or genetically-related pollen and prevent pollen tube growth soon after germination in order to avoid self-fertilization. Angiosperms have developed different reproductive barriers, one of the most extended being self-incompatibility (SI). With SI, pistils are able to reject self or genetically-related pollen thus promoting genetic variability. There are basically two distinct systems of SI: gametophytic (GSI) and sporophytic (SSI) based on their different molecular and genetic control mechanisms. In both types of SI, programmed cell death (PCD) has been found to play an important role in the rejection of self-incompatible pollen. Although reactive oxygen species (ROS) were initially recognized as toxic metabolic products, in recent years, a new role for ROS has become apparent: the control and regulation of biological processes such as growth, development, response to biotic and abiotic environmental stimuli, and PCD. Together with ROS, nitric oxide (NO) has become recognized as a key regulator of PCD. PCD is an important mechanism for the controlled elimination of targeted cells in both animals and plants. The major focus of this review is to discuss how ROS and NO control male-female cross-talk during fertilization in order to trigger PCD in self-incompatible pollen, providing a highly effective way to prevent self-fertilization.
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http://dx.doi.org/10.1093/jxb/erv083DOI Listing
May 2015

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana.

J Exp Bot 2015 May 5;66(9):2673-89. Epub 2015 Mar 5.

Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, 18008, Granada, Spain

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.
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http://dx.doi.org/10.1093/jxb/erv062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986871PMC
May 2015

Functional and structural changes in plant mitochondrial PrxII F caused by NO.

J Proteomics 2015 Apr 12;119:112-25. Epub 2015 Feb 12.

CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain. Electronic address:

Unlabelled: Peroxiredoxins (Prxs) have emerged as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events. Together with ROS, nitric oxide (NO) is a free radical product of the cell metabolism that is essential in the signal transduction. S-Nitrosylation is emerging as a fundamental protein modification for the transduction of NO bioactivity. Using recombinant pea mitochondrial PsPrxII F (PrxII F), the effect of S-nitrosoglutathione (GSNO) and sodium nitroprusside dehydrate (SNP), which are known to mediate protein S-nitrosylation processes, was studied. S-Nitrosylation of the PrxII F was demonstrated using the biotin switch method and LC ESI-QTOF tandem MS analysis. S-nitrosylated PrxII F decreased its peroxidase activity and acquired a new transnitrosylase activity, preventing the thermal aggregation of citrate synthase (CS). For the first time, we demonstrate the dual function for PrxII F as peroxidase and transnitrosylase. This switch was accompanied by a conformational change of the protein that could favor the protein-protein interaction CS-PrxII F. The observed in vivo S-nitrosylation of PrxII F could probably function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. We conclude that we are dealing with a novel regulatory mechanism for this protein by NO.

Biological Significance: S-Nitrosylation is a post-translational modification that is increasingly viewed as fundamental for the signal transduction role of NO in plants. This study demonstrates that S-nitrosylation of the mitochondrial peroxiredoxin PrxII F induces a conformational change in the protein and provokes a reduction in its peroxidase activity, while acquiring a novel function as transnitrosylase. The implication of this mechanism will increase our understanding of the role of posttranslational modifications in the protein function in plants under stress situations such as salinity, in which NO could act as signaling molecule.
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http://dx.doi.org/10.1016/j.jprot.2015.01.022DOI Listing
April 2015

Cadmium induces two waves of reactive oxygen species in Glycine max (L.) roots.

Plant Cell Environ 2014 Jul 24;37(7):1672-87. Epub 2014 Feb 24.

Laboratorio de Química Biológica. Departamento de Bioquímica y Ciencias Biológicas, FQByF. Universidad Nacional de San Luis, Ejercito de los Andes 950, San Luis, 5700, Argentina.

Cadmium (Cd) is a non-essential heavy metal that may be toxic or even lethal to plants as it can be easily taken up by the roots and loaded into the xylem to the leaves. Using soybean roots (Glycine max L.) DM 4800, we have analysed various parameters related to reactive oxygen metabolism and nitric oxide (NO) during a 6 day Cd exposure. A rise in H(2)O(2) and NO, and to a lesser extent O(2)(·-) content was observed after 6 h exposure with a concomitant increase in lipid peroxidation and carbonyl group content. Both oxidative markers were significantly reduced after 24 h. A second, higher wave of O(2)(·-) production was also observed after 72 h of exposure followed by a reduction until the end of the treatment. NOX and glicolate oxidase activity might be involved in the initial Cd-induced reactive oxygen species (ROS) production and it appears that other sources may also participate. The analysis of antioxidative enzymes showed an increase in glutathione-S-transferase activity and in transcript levels and activity of enzymes involved in the ascorbate-glutathione cycle and the NADPH-generating enzymes. These results suggest that soybean is able to respond rapidly to oxidative stress imposed by Cd by improving the availability of NADPH necessary for the ascorbate-glutathione cycle.
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http://dx.doi.org/10.1111/pce.12280DOI Listing
July 2014

Protein S-nitrosylation in plants under abiotic stress: an overview.

Front Plant Sci 2013 Sep 20;4:373. Epub 2013 Sep 20.

Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.

Abiotic stress is one of the main problems affecting agricultural losses, and understanding the mechanisms behind plant tolerance and stress response will help us to develop new means of strengthening fruitful agronomy. The mechanisms of plant stress response are complex. Data obtained by experimental procedures are sometimes contradictory, depending on the species, strength, and timing applied. In recent years nitric oxide has been identified as a key signaling molecule involved in most plant responses to abiotic stress, either indirectly through gene activation or interaction with reactive oxygen species and hormones; or else directly, as a result of modifying enzyme activities mainly by nitration and S-nitrosylation. While the functional relevance of the S-nitrosylation of certain proteins has been assessed in response to biotic stress, it has yet to be characterized under abiotic stress. Here, we review initial works about S-nitrosylation in response to abiotic stress to conclude with a brief overview, and discuss further perspectives to obtain a clear outlook of the relevance of S-nitrosylation in plant response to abiotic stress.
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http://dx.doi.org/10.3389/fpls.2013.00373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778396PMC
September 2013
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