Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant–microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca2+ involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca2+ chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca2+ influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed

Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant–microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca²⁺ involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the <i>nod</i> gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca²⁺ chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca²⁺ influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus -2

<p><b>Copyright information:</b></p><p>Taken from "Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus "</p><p>http://www.biomedcentral.com/1471-2229/7/41</p><p>BMC Plant Biology 2007;7():41-41.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950503.</p><p></p>from culture filtrates of (b), (c), and grown in the presence of (d). In c, the first peak of the Catransient induced by the >3 kDa metabolites is represented out of scale. In d, the inset shows the [Ca]changes induced by the simultaneous application of the metabolite fractions (>3 kDa,

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus -0

<p><b>Copyright information:</b></p><p>Taken from "Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus "</p><p>http://www.biomedcentral.com/1471-2229/7/41</p><p>BMC Plant Biology 2007;7():41-41.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950503.</p><p></p>from culture filtrates of (b), (c), and grown in the presence of (d). In c, the first peak of the Catransient induced by the >3 kDa metabolites is represented out of scale. In d, the inset shows the [Ca]changes induced by the simultaneous application of the metabolite fractions (>3 kDa,

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus -1

<p><b>Copyright information:</b></p><p>Taken from "Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus "</p><p>http://www.biomedcentral.com/1471-2229/7/41</p><p>BMC Plant Biology 2007;7():41-41.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1950503.</p><p></p> presence of (b)

Data_Sheet_1_The Rare Sugar Tagatose Differentially Inhibits the Growth of Phytophthora infestans and Phytophthora cinnamomi by Interfering With Mitochondrial Processes.PDF

Rare sugars are monosaccharides with limited availability in nature and their biological functions are largely unknown. Among them, tagatose was developed as a low-calorie sweetener and showed beneficial effects on human health. Tagatose is metabolized by only certain microbial taxa and inhibits the growth of important crop pathogens (e.g., Phytophthora infestans), but its mode of action and the microbial responses are unknown. The aim of this study was to understand the tagatose mode of action against Phytophthora spp., with the final aim of developing new plant protection products. Tagatose inhibited P. infestans growth in vitro and caused severe ultrastructural alterations, with the formation of circular and concentric mitochondrial cristae. Decreased ATP content and reduced oxygen consumption rate (OCR) were found in tagatose-incubated P. infestans as compared to the control, with the consequent accumulation of reactive oxygen species (ROS) and induction of genes related to apoptosis and oxidative stress response. On the other hand, tagatose did not, or only slightly, affect the growth, cellular ultrastructure and mitochondrial processes in Phytophthora cinnamomi, indicating a species-specific response to this rare sugar. The mode of action of tagatose against P. infestans was mainly based on the inhibition of mitochondrial processes and this rare sugar seems to be a promising active substance for the further development of eco-friendly fungicides, thanks to its anti-nutritional properties on some phytopathogens and low risk for human health.</p

Image_5_Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.png

IntroductionChloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.MethodsIn the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress.ResultsAltogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. ConclusionsThese observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.</p

Image_4_Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.tif

IntroductionChloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.MethodsIn the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress.ResultsAltogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. ConclusionsThese observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.</p

Table_9_Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.xlsx

IntroductionChloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.MethodsIn the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress.ResultsAltogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. ConclusionsThese observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.</p

Table_2_Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.docx

IntroductionChloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle.MethodsIn the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress.ResultsAltogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. ConclusionsThese observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.</p