Oxidative Signaling

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H2O2) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mM H2O2 on several oxidative features such as protein carbonylation, endogenous H2O2 and lipid peroxidation levels. We report that H2O2 treatment of the pea seeds increased their endogenous H2O2 content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H2O2 in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H2O2, whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H2O2 in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death...

In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death and oxidative damage in response to ultraviolet radiation. Complementation of CRK5 mutation managed to recover the wild-type phenotype, indicating an essential role of this gene in the regulation of growth, development, and acclimatory responses.

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H2O2) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mM H2O2 on several oxidative features such as protein carbonylation, endogenous H2O2 and lipid peroxidation levels. We report that H2O2 treatment of the pea seeds increased their endogenous H2O2 content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H2O2 in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H2O2, whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H2O2 in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H2O2) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mM H2O2 on several oxidative features such as protein carbonylation, endogenous H2O2 and lipid peroxidation levels. We report that H2O2 treatment of the pea seeds increased their endogenous H2O2 content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H2O2 in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H2O2, whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H2O2 in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

Hydrogen peroxide (H 2 O 2) operates as a signaling molecule in eukaryotes, but the specificity of its signaling capacities remains largely unrevealed. Here, we analyzed whether a moderate production of H 2 O 2 from two different plant cellular compartments has divergent effects on the plant transcriptome. Arabidopsis thaliana overexpressing glycolate oxidase in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012) and plants deficient in peroxisomal catalase (Queval et al., 2007; Inzé et al., 2012) were grown under non-photorespiratory conditions and then transferred to photorespiratory conditions to foster the production of H 2 O 2 in both organelles. We show that H 2 O 2 originating in a specific organelle induces two types of responses: one that integrates signals independently from the subcellular site of H 2 O 2 production and another that is dependent on the H 2 O 2 production site. H 2 O 2 produced in peroxisomes induces transcripts involved in protein repair responses, while H 2 O 2 produced in chloroplasts induces early signaling responses, including transcription factors and biosynthetic genes involved in production of secondary signaling messengers. There is a significant bias towards the induction of genes involved in responses to wounding and pathogen attack by chloroplastic-produced H 2 O 2 , including indolic glucosinolates-, camalexin-, and stigmasterol-biosynthetic genes. These transcriptional responses were accompanied by the accumulation of 4-methoxy-indol-3-ylmethyl glucosinolate and stigmasterol.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics |

Abiotic stress, including drought, cold, high temperature and UV radiation, produce common responses in plant tissue, resulting in the overproduction of Reactive Oxygen Species (ROS). ROS molecules damage cell membranes and disrupt plant metabolism. Our previous experiments indicated that naturally chelated/complexed foliar nutritional supplement products reduce the amount of ROS molecules formed under stress conditions. In this study, we investigated the short-term effect of the novel proprietary biostimulant CYT31 on gene expression in plants exposed to drought using Arabidopsis thaliana grown under controlled conditions. Plants were sprayed with biostimulant CYT31 (treated) or water (control). One hour after spraying, plants were exposed to drought conditions for one, two or four hours. Tissue was collected for two-color microarray and cell damage analysis. Tissue damage was evaluated using an electrolyte leakage-detection method. Drought stress up-regulated over 245 genes involved in water deprivation, redox and stress response. The gene response was influenced by the severity of the stress and correlated with tissue damage. The up-regulation of these genes was significantly altered in the tissue treated with biostimulant CYT31, suggesting the activation of several mechanisms leading to the reduction of tissue injury. Gene activation was confirmed by quantitative polymerase chain reaction (qPCR) analysis. This paper describes a proposed model for metabolic pathways activated by biostimulant CYT31 in plants exposed to drought stress.

Hydrogen peroxide (H 2 O 2) operates as a signaling molecule in eukaryotes, but the specificity of its signaling capacities remains largely unrevealed. Here, we analyzed whether a moderate production of H 2 O 2 from two different plant cellular compartments has divergent effects on the plant transcriptome. Arabidopsis thaliana overexpressing glycolate oxidase in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012) and plants deficient in peroxisomal catalase (Queval et al., 2007; Inzé et al., 2012) were grown under non-photorespiratory conditions and then transferred to photorespiratory conditions to foster the production of H 2 O 2 in both organelles. We show that H 2 O 2 originating in a specific organelle induces two types of responses: one that integrates signals independently from the subcellular site of H 2 O 2 production and another that is dependent on the H 2 O 2 production site. H 2 O 2 produced in peroxisomes induces transcripts involved in protein repair responses, while H 2 O 2 produced in chloroplasts induces early signaling responses, including transcription factors and biosynthetic genes involved in production of secondary signaling messengers. There is a significant bias towards the induction of genes involved in responses to wounding and pathogen attack by chloroplastic-produced H 2 O 2 , including indolic glucosinolates-, camalexin-, and stigmasterol-biosynthetic genes. These transcriptional responses were accompanied by the accumulation of 4-methoxy-indol-3-ylmethyl glucosinolate and stigmasterol.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics |

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics |

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics |

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H2O2) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mM H2O2 on several oxidative features such as protein carbonylation, endogenous H2O2 and lipid peroxidation levels. We report that H2O2 treatment of the pea seeds increased their endogenous H2O2 content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H2O2 in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H2O2, whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H2O2 in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

In a previous publication, we showed that the treatment of
pea seeds in the presence of hydrogen peroxide (H2O2)
increased germination performance as well as seedling
growth.To gain insight into the mechanisms responsible for
this behaviour, we have analysed the effect of treating
mature pea seeds in the presence of 20 mM H2O2 on several
oxidative features such as protein carbonylation, endogenous
H2O2 and lipid peroxidation levels. We report that
H2O2 treatment of the pea seeds increased their endogenous
H2O2 content and caused carbonylation of storage
proteins and of several metabolic enzymes.Under the same
conditions,we also monitored the expression of two MAPK
genes known to be activated by H2O2 in adult pea plants.
The expression of one of them, PsMAPK2, largely
increased upon pea seed imbibition in H2O2, whereas no
change could be observed in expression of the other,
PsMAPK3. The levels of several phytohormones such as
1-aminocyclopropane carboxylic acid, indole-3-acetic acid
and zeatin appeared to correlate with the measured oxidative
indicators and with the expression of PsMAPK2. Globally,
our results suggest a key role of H2O2 in the
coordination of pea seed germination, acting as a priming
factor that involves specific changes at the proteome, transcriptome and hormonal levels.

Hydrogen peroxide (H2O2) increased the germination percentage
of pea seeds, as well as the growth of seedlings in a
concentration-dependent manner. The effect of H2O2 on
seedling growth was removed by incubation with 10 mM
ABA. The H2O2-pretreatment produced an increase in
ascorbate peroxidase (APX), peroxidase (POX) and
ascorbate oxidase (AAO).The increases in these ascorbateoxidizing
enzymes correlated with the increase in the
growth of the pea seedlings as well as with the decrease in
the redox state of ascorbate.Moreover, the increase inAPX
activity was due to increases in the transcript levels of cytosolic
and stromal APX (cytAPX, stAPX).
The proteomic analysis showed that H2O2 induced proteins
related to plant signalling and development, cell elongation
and division, and cell cycle control.
A strong correlation between the effect of H2O2 on plant
growth and the decreases in ABA and zeatin riboside (ZR)
was observed.
The results suggest an interaction among the redox state
and plant hormones, orchestrated by H2O2, in the induction
of proteins related to plant signalling and development
during the early growth of pea seedlings.

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by
the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The
CRKs form one of the largest groups of receptor-like protein kinases in plants, but their bio-
logical functions have so far remained largely uncharacterized. We conducted a large-scale
phenotyping approach of a nearly complete
crk
T-DNA insertion line collection showing that
CRKs control important aspects of plant development and stress adaptation in response to
biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aper-
ture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play gen-
eral and fine-tuning roles in the regulation of stomatal closure induced by microbial and
abiotic cues. Despite their great number and high similarity, large-scale phenotyping identi-
fied specific functions in diverse processes for many CRKs and indicated that CRK2 and
CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a
whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct
responses in an antagonistic fashion suggesting future potential for using CRKs in genetic
approaches to improve plant performance and stress tolerance.

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during afterripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics | oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive PLOS Genetics | oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.