Ana Villasuso - Academia.edu (original) (raw)

Papers by Ana Villasuso

Environmental and Experimental Botany, Oct 1, 2019

Fusarium is the major causal agent of Fusarium head blight (FHB) in several cereal crops. Macroco... more Fusarium is the major causal agent of Fusarium head blight (FHB) in several cereal crops. Macroconidia, the asexual form of Fusarium, can infect the roots and aerial parts of plants. The response of cereal to attacks by Fusarium has been investigated at transcriptomic and metabolomic level, but there are no reports of root lipidomic assays carried out during Fusarium-root interaction. In order to determine how root phospholipids contribute to that interaction, we performed a lipidomics-based ESI-MS/MS assay coupled with a statistical analysis. It was found that phospholipid and galactolipid levels were not modified during early pathogen response, although some individual phospholipid molecular species did undergo significant changes. In plants exposed to macroconidia there was a rapid and transient increase in the phosphatidylcholine (36:4-PC) molecular species, in comparison with the control. By contrast, there was a decrease in lysophosphatidylcholine (16:0-, 18:2-and 18:3-LPC) levels. Furthermore, a phospholipase assay and the measurement of endogenous phytohormone levels through the use of fluorescent lipids and liquid chromatography-tandem mass spectrometry, revealed an increase in phospholipase A (PLA) activity, as well as in the endogenous amounts of Jasmonic acid (JA) and Salicylic acid (SA). The results indicate that barley root is able to modulate the glycerolipid fatty acid composition during the early response to Fusarium, suggesting a particular pathogen-sensing mechanism that could be useful to understand the role of lipids in plant-pathogen interactions.

Biochemistry and Molecular Biology Education

Plant Physiology and Biochemistry, 2016

The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid... more The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

Environmental and Experimental Botany, 2016

Abstract Vicia sativa, is a leguminous species able to germinate, grow in the presence of phenol ... more Abstract Vicia sativa, is a leguminous species able to germinate, grow in the presence of phenol and remove this contaminant. However, there are not reports concerning the signals triggered by the pollutant and how plants perceive and transduce this signal in order to adapt to adverse conditions. Phosphatidic acid (PA) has been proposed as a key messenger in plants and it can be generated via phospholipase D (PLD) or via phospholipase C (PLC) coupled to diacylglycerol kinase (DGK). Thus, changes in this minor phospholipid and in enzymes involved in its catabolism were analyzed after treatment with phenol (25 and 100 mg L−1). The results obtained, seem to suggest that the higher concentration could be sensed as a stressful signal, since a rapid (1.5 h) and transient increase in PA, via PLD and a second wave of increase possibly via PLC/DGK was observed after 96 h of exposure with 100 mg L−1 of phenol. Besides, a markedly increase in enzymes related with PA metabolism, mainly DGK, phosphatidylinositol kinase (PIK) and PA kinase (PAK), was detected after long term treatment. Thus, this study highlighted the key role of minor phospholipids, especially PA, in the transduction pathway induced by phenol.

Plant Physiology and Biochemistry, 2012

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone ... more We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Plant Physiology and Biochemistry, 2013

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (G... more Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [ 32 P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase e that allows the production of 18:2-DGPP from 18:2-PA e is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

Lipids, 2011

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in ... more Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005

Physiological and cellular adaptations to environmental changes are known to be related to modifi... more Physiological and cellular adaptations to environmental changes are known to be related to modifications in membrane lipids. This work provides metabolic and compositional evidence that Trypanosoma cruzi epimastigotes are able to synthesize and desaturate fatty acids, to incorporate them into their lipids, and to modify this incorporation when carbamoylcholine is present in the medium. The fatty acids formed from [2-14 C]acetate in the period from 2 to 9 days were mostly (70%) incorporated in phospholipids, the remainder 30% being recovered in neutral lipids, such as triacylglycerols (TAG) and diacylglycerols (DAG). The main fatty acids formed from [2-14 C]acetate were saturates (16:0, 18:0), monoenes (16:1, 18:1) and dienes (mostly 18:2). The ratios between labelled unsaturated and saturated fatty acids increased continuously with growth, consistent with a precursor-product relationship between the main fatty acids, and with the occurrence in T. cruzi of D 9-and D 12-desaturases. From days 2 to 5, [ 14 C]18:2 was the main fatty acid produced. Accordingly, the fatty acid profiles showed a significant increase in the percentage of 18:2 in all lipids in the period under study, especially in the first 2 to 5 days. In the presence of carbamoylcholine, the labelling of DAG and TAG with [ 14 C]18:2 augmented. The results indicate that T cruzi is able to synthesize the main types of fatty acids required to form its membrane lipids, and to exchange them actively in response to environmental stimuli.

Physiologia Plantarum, 2008

ABA plays an important regulatory role in seed germination because it inhibits the response to GA... more ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of a-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg 2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone.

Plant Physiology and Biochemistry

Environmental Science and Pollution Research

Chemistry and Physics of Lipids

Environmental and Experimental Botany

Biochemistry and Molecular Biology Education

Plant Physiology and Biochemistry

Environmental and Experimental Botany

Environmental and Experimental Botany, Oct 1, 2019

Fusarium is the major causal agent of Fusarium head blight (FHB) in several cereal crops. Macroco... more Fusarium is the major causal agent of Fusarium head blight (FHB) in several cereal crops. Macroconidia, the asexual form of Fusarium, can infect the roots and aerial parts of plants. The response of cereal to attacks by Fusarium has been investigated at transcriptomic and metabolomic level, but there are no reports of root lipidomic assays carried out during Fusarium-root interaction. In order to determine how root phospholipids contribute to that interaction, we performed a lipidomics-based ESI-MS/MS assay coupled with a statistical analysis. It was found that phospholipid and galactolipid levels were not modified during early pathogen response, although some individual phospholipid molecular species did undergo significant changes. In plants exposed to macroconidia there was a rapid and transient increase in the phosphatidylcholine (36:4-PC) molecular species, in comparison with the control. By contrast, there was a decrease in lysophosphatidylcholine (16:0-, 18:2-and 18:3-LPC) levels. Furthermore, a phospholipase assay and the measurement of endogenous phytohormone levels through the use of fluorescent lipids and liquid chromatography-tandem mass spectrometry, revealed an increase in phospholipase A (PLA) activity, as well as in the endogenous amounts of Jasmonic acid (JA) and Salicylic acid (SA). The results indicate that barley root is able to modulate the glycerolipid fatty acid composition during the early response to Fusarium, suggesting a particular pathogen-sensing mechanism that could be useful to understand the role of lipids in plant-pathogen interactions.

Biochemistry and Molecular Biology Education

Plant Physiology and Biochemistry, 2016

The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid... more The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

Environmental and Experimental Botany, 2016

Abstract Vicia sativa, is a leguminous species able to germinate, grow in the presence of phenol ... more Abstract Vicia sativa, is a leguminous species able to germinate, grow in the presence of phenol and remove this contaminant. However, there are not reports concerning the signals triggered by the pollutant and how plants perceive and transduce this signal in order to adapt to adverse conditions. Phosphatidic acid (PA) has been proposed as a key messenger in plants and it can be generated via phospholipase D (PLD) or via phospholipase C (PLC) coupled to diacylglycerol kinase (DGK). Thus, changes in this minor phospholipid and in enzymes involved in its catabolism were analyzed after treatment with phenol (25 and 100 mg L−1). The results obtained, seem to suggest that the higher concentration could be sensed as a stressful signal, since a rapid (1.5 h) and transient increase in PA, via PLD and a second wave of increase possibly via PLC/DGK was observed after 96 h of exposure with 100 mg L−1 of phenol. Besides, a markedly increase in enzymes related with PA metabolism, mainly DGK, phosphatidylinositol kinase (PIK) and PA kinase (PAK), was detected after long term treatment. Thus, this study highlighted the key role of minor phospholipids, especially PA, in the transduction pathway induced by phenol.

Plant Physiology and Biochemistry, 2012

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone ... more We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Plant Physiology and Biochemistry, 2013

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (G... more Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [ 32 P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase e that allows the production of 18:2-DGPP from 18:2-PA e is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

Lipids, 2011

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in ... more Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005

Physiological and cellular adaptations to environmental changes are known to be related to modifi... more Physiological and cellular adaptations to environmental changes are known to be related to modifications in membrane lipids. This work provides metabolic and compositional evidence that Trypanosoma cruzi epimastigotes are able to synthesize and desaturate fatty acids, to incorporate them into their lipids, and to modify this incorporation when carbamoylcholine is present in the medium. The fatty acids formed from [2-14 C]acetate in the period from 2 to 9 days were mostly (70%) incorporated in phospholipids, the remainder 30% being recovered in neutral lipids, such as triacylglycerols (TAG) and diacylglycerols (DAG). The main fatty acids formed from [2-14 C]acetate were saturates (16:0, 18:0), monoenes (16:1, 18:1) and dienes (mostly 18:2). The ratios between labelled unsaturated and saturated fatty acids increased continuously with growth, consistent with a precursor-product relationship between the main fatty acids, and with the occurrence in T. cruzi of D 9-and D 12-desaturases. From days 2 to 5, [ 14 C]18:2 was the main fatty acid produced. Accordingly, the fatty acid profiles showed a significant increase in the percentage of 18:2 in all lipids in the period under study, especially in the first 2 to 5 days. In the presence of carbamoylcholine, the labelling of DAG and TAG with [ 14 C]18:2 augmented. The results indicate that T cruzi is able to synthesize the main types of fatty acids required to form its membrane lipids, and to exchange them actively in response to environmental stimuli.

Physiologia Plantarum, 2008

ABA plays an important regulatory role in seed germination because it inhibits the response to GA... more ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of a-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg 2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone.

Plant Physiology and Biochemistry

Environmental Science and Pollution Research

Chemistry and Physics of Lipids

Environmental and Experimental Botany

Biochemistry and Molecular Biology Education

Plant Physiology and Biochemistry

Environmental and Experimental Botany