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Ion fluxes and other second messengers

As indicated earlier, the depolarization of the plasma membrane causes ion fluxes, the most important being Ca2+ ions as they have been shown to function as second messengers in many signaling pathways (Kudla et al., 2010). Plants can discriminate between various stimuli based on calcium signatures that are defined by their amplitude, frequency, subcellular localization and duration. Peaks of cytosolic Ca2+ are detected within 6 seconds in cells close to the injury site (Beneloujaephajri et al., 2013) and several studies showed that cytosolic calcium levels also increase upon herbivore attack (Arimura & Maffei, 2010). Very recently, the use of a fluorescent calcium biosensor has allowed observing elevations of Ca2+ levels during a biotic interaction with an aphid (Myzus persicae) (Vincent et al., 2017). Using appropriate mutant lines, authors were able to show that these Ca2+ elevations are dependent on BAK1 (Brassinosteroid insensitive-Associated Kinase 1), a well-known co-receptor of Pathogen Recognition Receptors (PRRs), as well as on GLR3.3 and 3.6 channels (GLutamate Receptor-like). In the same study, the vacuolar cation channel TPC1 (Two Pore Channel 1) was shown to importantly contribute to the observed cytosolic elevation. In another study, TPC1-dependent calcium elevations were observed upon feeding of the caterpillar Spodoptera littoralis, both locally and systemically (Kiep et al., 2015). Inositol trisphosphate (InsP3) was shown to mediate Ca2+ release from internal stores (Alexandre & Lassalles, 1990). It was recently shown, along with phosphoinositide, to act as a second messenger of wounding signal transduction and regulate the expression of wound-inducible genes (Mosblech et al., 2008). Interestingly, their production was shown to require the biosynthesis of JA as no increase of InsP3 was observed in plants deficient in JA synthesis.
In Arabidopsis, the calcium signal is decoded by three major multigenic families of Ca2+ sensor proteins: calmodulins or calmodulin-like proteins (CAMs), calcineurin B-like proteins (CBLs) and Calcium-Dependent Protein Kinases (CDPKs). Some of those sensors have been studied and were shown to have roles in plant immunity in response to bacterial pathogens but also herbivorous insects (Boudsocq & Sheen, 2013; Arimura & Maffei, 2010). In Arabidopsis, some CDPKs are activated by the bacterial elicitor flg22 and were shown to be important for the transduction of defense responses (Boudsocq et al., 2010). Two CDPKs, CPK3 and CPK13, were directly shown to act as positive regulators of the defense gene PDF1.2 upon exposure to Spodoptera littoralis caterpillars (Kanchiswamy et al., 2010). A calmodulin-like protein, CML42 was shown to sense Ca2+ upon application of oral secretions from S. littoralis and to connect Ca2+ to JA signaling (Vadassery et al., 2012). cml42 plants showed enhanced resistance to the caterpillar as well as higher expression levels of the JA-responsive gene VSP2. Another calmodulin-like protein, CML37, was shown to be involved in the regulation of the Jasmonic Acid (JA) pathway and in resistance to herbivory (Scholz et al., 2014). Three TCH (TouCH) genes encoding calmodulin and calmodulin-like proteins were shown to be induced by mechanical stimuli such as touch or wind, supporting the important role of calcium signaling in the transduction of such environmental cues (Braam & Davis, 1990). The role of Ca2+ sensor proteins in a context of wounding and/or herbivory was also demonstrated in other plants species. Thus, in tomato, LeCDPK2 is involved in the wound-induced ethylene (ET) production (Kamiyoshihara et al., 2010) and in tobacco (Nicotiana attenuata), NaCDPK4 and NaCDPK5 negatively regulate wounding responses and resistance to the herbivore Manduca sexta (Yang et al., 2012). Finally, in maize, ZmCPK11 is activated by wounding in a JA-dependent pathway and is involved in systemic wound responses (Szczegielniak et al., 2012). In addition to calcium signaling, phospholipase D (PLD) has been suggested to play an important role in the mediation of wound-induced responses (Wang et al., 2000). PLD hydrolyzes phospholipids and leads to the production of phosphatidic acid (PA). PLD-induced hydrolysis was observed after wounding in bean leaves and appeared to be mediated by an increase in cytoplasmic Ca2+ concentrations (Ryu & Wang, 1996). Interestingly, PLD was shown to be required for the production of JA and the induction of a JA-regulated gene (Wang et al., 2000; Bargmann et al., 2009).

Production of Reactive Oxygen Species (ROS)

Reactive Oxygen Species (ROS) are normally produced in peroxisomes, chloroplasts and mitochondria due to the presence of an oxidizing activity or electron transfer chains in these organelles (Tripathy & Oelmuller, 2012). In addition, ROS production can be induced upon stress conditions and create a ROS burst that is thought to have antimicrobial effects. Stress-induced ROS production was first described in 1983 in potato tubers infected with the oomycete Phytophthora infestans (Doke, 1983). Since this study, many works contributed to show that ROS can also be involved in other stresses including wounding and insect feeding (Suzuki & Mittler, 2012; Maffei et al., 2006).
NADPH oxidases are well known producers of ROS (Marino et al., 2012). They transfer electrons from cytosolic NADPH (or NADH) to oxygen in the apoplast, thus leading to the production of superoxide ions (O2-). The latter is then converted to hydrogen peroxide (H2O2) in the apoplast by the action of superoxide dismutase. NADPH oxidases belong to the Respiratory Burst Oxidase Homolog (RBOH) family that has 10 members (RBOHA to RBOHJ) in Arabidopsis (Torres & Dangl, 2005). In response to wounding, the observed oxidative burst is believed to be due to NADPH oxidases. Indeed, in Arabidopsis seedlings, the rapid local and systemic ROS burst is impaired in rbohd mutants (Miller et al., 2009). In tomato (Solanum lycopersicum), the inhibition of NADPH oxidase by specific inhibitors blocks the wound-induced production of ROS and the induction of some defense genes. NADPH oxidases are also involved in wound-induced ROS burst in other plant species such as Medicago truncatula or potato (Soares et al., 2011; Bernards & Razem, 2001). In addition to their oxidase domain, NADPH oxidases also possess Ca2+ binding domains and it was demonstrated that wound-induced ROS burst is dependent on Ca2+ peaks (Monshausen et al., 2009; Beneloujaephajri et al., 2013). Besides NADPH oxidases, ROS can also be produced through the action of apoplastic peroxidases but no evidence was found for their action in wound-induced ROS production in Arabidopsis (Minibayeva et al., 2014). Concerning herbivory, it has been shown that H2O2 is released upon Spodoptera littoralis feeding on Lima bean and, to a lesser extent, upon mechanical damage (Maffei et al., 2006).

Damage-Associated Molecular Patterns (DAMPs) are intercellular signaling molecules produced upon wounding

Plants are able to sense their damaged self and trigger responses very similar to those induced by pathogen infection. Endogenous molecules deriving from wounded tissue can act like Damage-Associated Molecular Patterns (DAMPs) and elicit local and systemic plant defenses. In tomato, systemin, an 18 amino-acid peptide, is released in the apoplast after wounding or insect attack (Jacinto et al., 1997). Sensing of systemin leads to the biosynthesis of JA, an important wound phytohormone, that activate defense responses locally and in the neighboring cells (Orozco-Cardenas et al., 1993). Hydroxyproline-rich systemins have also been identified in other Solanaceae (potato, petunia, black nightshade) and are able to trigger plant immunity in response to herbivore attack. In Arabidopsis thaliana, three self-derived families of molecules have been identified so far. They are all recognized by Pattern- Recognition Receptors (PRRs) localized at the plasma membrane level. Oligogalacturonides (OGs) are released from the plant cell wall after degradation of homogalacturonan, the main component of pectin, through the action of wounding-induced hydrolytic enzymes such as polygalacturonase. The size of OGs is important and can determine their efficiency as eliciting molecules. OGs with a degree of polymerization between 10 and 15 are the most active ones (Ferrari et al., 2013). Wall-Associated Kinases (WAKs) are RLKs associated to the cell wall that are required for cell expansion during development but can also mediate stress responses induced by OGs released upon wounding. WAKs are encoded by 5 genes among which WAK1 and WAK2 are transcriptionally induced by wounding. In rice, OsWAK1 is also upregulated by wounding as well as salicylic acid (SA) and JA. By the use of a chimeric receptor approach, authors have shown the ability of WAK1 to bind OGs (Brutus et al., 2010). WAK2 also seems to have a predominant role as wak2-1 plants are not able to activate defense responses induced by OGs (Kohorn et al., 2009). Adenosine Tri Phosphate (ATP) can also act as an extracellular signaling molecule (Tanaka et al., 2014). It is released from plant cells in response to abiotic stresses (Dark et al., 2011), fungal elicitors and mechanical stimuli (Weerasinghe et al., 2009). Concentrations of ATP up to 40 µM were measured after wounding, probably released from neighboring cells whose plasma membrane had lost its integrity (Jeter et al., 2004). Recently the plant receptor of ATP has been identified in Arabidopsis as DORN1 (DOes not Respond to Nucleotides 1), a lectin receptor kinase (Choi et al., 2014). The characterization of dorn1 plants showed the specificity of this receptor for ATP as plants were not impaired in responses to other exogenous stimuli. Based on this observation, transcriptional responses to ATP were compared to those observed upon wounding. 60% of the genes induced by ATP were also induced by wounding and the expression of selected genes was found highly reduced in dorn1 plants after wounding, strongly suggesting that ATP is a major component of wounding response.

JA homeostasis in response to wounding and herbivory

Jasmonates form a family of oxylipins deriving from enzymatic oxygenation of 18 and 16-carbon triunsaturated fatty acids (Wasternack & Kombrink, 2010). JA is synthesized from the fatty acid α-linolenic acid (18:3) (α-LeA) released from galactolipids of chloroplast membranes (figure 1.2). The enzyme responsible for the release of α-LeA was identified as phospholipase 1 (PLA1). α-LeA is then oxidized by a lipoxygenase (LOX) that is responsible for the insertion of an oxygen at the C-13 position of the acid, thus forming 13-HPOT. The genome of Arabidopsis includes 4 LOXs involved in JA synthesis upon wounding (LOX2, 3, 4 and 6). LOX2 was shown to be required for JA production proximal to the wound (Glauser et al., 2009). However, JA and JA-Ile are still synthesized in the lox2-1 mutant suggesting the involvement of other LOXs. More recent studies using combinations of multiple LOXs mutants revealed that all 4 LOXs contribute to JA synthesis upon wounding (Chauvin et al., 2013). LOX6 was found to have a particular role in early JA synthesis in distal leaves (Farmer et al., 2014). The next step of the JA biosynthesis pathway leads to the formation of its precursor cis-12-oxo-phytodienoic acid (OPDA). The enzymes responsible for this step are ALLENE OXIDE CYCLASE (AOC) and ALLENE OXIDE SYNTHASE (AOS). OPDA is then transported to the peroxisome where it is reduced to OPC-8:0 by 12-oxophytodienoate reductase (OPR3). Next, (+)-7-iso-JA is produced after several rounds of β-oxidations. Upon its subsequent transport to the cytoplasm, JA is modified to methyl-JA (meJA), JA-Ile or other derivatives. JA-Ile is formed by the action of the JAR1 enzyme (Jasmonic Acid Resistant 1) and is the most active form of JA as it seems to be the one that actually plays the major role in Arabidopsis leaves (Staswick & Tiryaki, 2004; Fonseca et al., 2009b).
At the histological level, jasmonate biosynthesis in Arabidopsis mainly takes place in the vascular bundles of the vegetative parts of the plant. However, some data suggest a production of JA in cells outside the vessels. For example, LOX2 is found strongly expressed in mesophyll cells (Montillet et al., 2013). The use of promoter::GUS lines allowed the observation of some genes encoding enzymes of the AOC family in Arabidopsis roots (Stenzel et al., 2012).
The Arabidopsis microarray datasets from various stress conditions and developmental stages revealed transcriptional regulation of all JA biosynthesis genes (Pauwels et al., 2009; van Verk et al., 2011). However, the activity of some enzymes seems to be post-translationally regulated. Thus, the activity of OPR3 seems to result from a monomer/dimer equilibrium (Schaller & Stintzi, 2009). Unstressed leaves of Arabidopsis contain very low amounts of bioactive JA, typically about 20-50 pmol/g fresh mass but this level increases within 5 minutes upon wounding (Glauser et al., 2008b; Glauser et al., 2009). In the wounded leaf JA levels increase up to 500-fold, reaching about 10 nmol/g fresh mass whereas JA-Ile levels reach less than 10% of the level of JA (Suza & Staswick, 2008). In more details, the first significant increase of JA in the wounded leaf is observed less than 30 seconds after wounding and its level doubles every 20 seconds during the first minute. Concerning JA-Ile, its first significant increase is observed 5 min after wounding (Koo et al., 2009). This remarkable speed of production suggests that all biosynthetic enzymes involved in production of JA/JA-Ile are already present in resting cells.
The molecular events linking wounding to the de novo synthesis of JA are largely unknown. Genetic and pharmacological disruption of cell wall integrity has been shown to constitutively activate the JA pathway (Hamann et al., 2009). There is also evidence suggesting that JA/JA-Ile are produced upon perception of DAMPs or bacterial effectors (Campos et al., 2014). In Arabidopsis, the perception of AtPep1 by its receptors PEPR1 and PEPR2 was shown to have a role in JA production as pepr1 pepr2 plants accumulated less JA upon herbivore oral secretion (Klauser et al., 2015). In maize, ZmPEPR3 activates JA synthesis by increasing the level of transcripts encoding the biosynthetic enzymes AOC and AOS (Huffaker et al., 2006). The application of ATP induces the expression of genes encoding enzymes for JA biosynthesis in Arabidopsis (Song et al., 2006; Choi et al., 2014). The responses induced by OGs were however shown to be independent from JA signaling (Ferrari et al., 2007). The exact mechanism by which DAMP signaling leads to JA biosynthesis still remains elusive. Among the intracellular events that could link the perception of self-derived elicitors to JA production are Ca2+ ions, ROS, MAPK cascades and CDPKs (Arimura & Maffei, 2010; Zebelo & Maffei, 2015). For instance, Ca2+ fluxes and associated CDPKs exert a control during the activation of JA biosynthesis genes (Bonaventure et al., 2007). However, it is not clear whether any of the enzymes involved in the biosynthesis of JA are regulated by CDPK/MAPK-dependent phosphorylation or cellular redox changes, although JA-induced phosphorylation of JA signaling components has been observed (Zhai et al., 2013).
In addition to JA-Ile, diverse conjugated and oxidized derivatives of JA are produced. These forms of jasmonates include hydroxyhasmonates (HOJAs) and HOJA-Ile as well as dicarboxyjasmonate (HOOCJA-Ile). HOJAs and HOOCJA-Ile accumulate in midveins of wounded leaves (Glauser et al., 2008) and were shown to be products of JA catabolism (Heitz et al., 2012; Koo et al., 2012). These derivatives being less active than JA-Ile in promoting the binding of COI1 to JAZ proteins, they participate to the attenuation of JA responses. Enzymes involved in JA-Ile catabolism are also rapidly induced upon wounding, herbivory and exogenous JA treatments (Bhosale et al., 2013). This co-expression of catabolism genes with other JA-response genes reflects the tight control of JA signaling.

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JA signaling through COI1 -JAZ- MYC signaling module

In 1994, the first JA resistant mutant coi1 (coronatine insensitive 1) was isolated and shown to be  impaired in the receptor of JA-Ile (Feys et al., 1994). In Arabidopsis, plants impaired in COI1 do not respond to external JA application. In addition, biochemical approaches showed direct binding of JA-Ile (or its structural mimic coronatine) to COI1 (Yan et al., 2009). COI1 is an F-box protein containing 18 leucine-rich repeat (LRR) that forms a complex with SCF (Skp1/Cullin/F-box), an E3 ubiquitin ligase which mediates protein ubiquitination for targeted degradation by the 26S proteasome (Xie et al., 1998). SCF/COI1 defines a complex specifically involved in jasmonate signaling (Xu et al., 2002). Mutants lacking a functional COI1 are compromised in all known JA-regulated processes and do not respond to exogenous JA, as shown in tobacco, tomato and Arabidopsis (Paschold et al., 2007; Li et al., 2004; Xie et al., 1998). Interestingly, the accumulation of JA-Ile was shown to be dependent on COI1 as wound-induced JA levels were far lower in coi1 plants than in wild-type ones (Chung et al., 2008). Jasmonate ZIM-Domain (JAZ) proteins that form a family of 13 members in Arabidopsis are negative regulators of the transcription of JA-responsive genes (Thines et al., 2007). The absence of DNA-binding domains in JAZ proteins suggests a) In the absence of JA-Ile, the repressor JAZ is bound to transcription factors of the MYC family, thus restraining their action.
b) When JA-Ile is produced in necessary amounts, it binds to the SCF/COI1 complex, allowing the latter to recruit JAZ repressors and promote their ubiquitination for further degradation by the proteasome. MYC factors are then set free and can activate the expression of defense genes. that the negative regulation is probably not performed by direct binding to gene promoter sequences but rather by interfering with transcription factors. Indeed, JAZ proteins can bind to many transcription factors and repress their activity. Among them the basic helix-loop-helix (bHLH) transcription factors MYC2, MYC3 and MYC4 have been extensively studied (Fernandez-Calvo et al., 2011). In the absence of JA-Ile, JAZ proteins are stable and repress the action of MYCs, and thus the expression of the downstream MYC-dependent genes, by recruiting two co-repressors TPL (TOPLESS) and NINJA (NOVEL INTERACTOR OF JAZ) and by competing with MED25 (MEDIATOR 25) for interaction with MYCs (figure 1.3). The myc2 myc3 myc4 triple mutant was shown to be highly susceptible to Spodoptera littoralis feeding (Schweizer et al., 2013). JA-Ile binding to COI1 promotes ubiquitination of JAZ proteins and their subsequent degradation by proteolysis (Chini et al., 2007). Protein-protein interaction assays showed that the COI1-JAZ interaction is stimulated by JA-Ile but not by meJA or OPDA (Thines et al., 2007). Poly-ubiquitination of JAZ proteins has been indirectly observed using proteomic approaches (Nagels et al., 2016) and their proteasome-dependent degradation was demonstrated by the use of MG132 treatments that stabilized JAZ proteins (Thines et al., 2007).
Interestingly, the expression of JAZ repressors is induced upon wounding and herbivory (Chung et al., 2008). More particularly, the JAZ10 gene is a well-described early marker for JA signaling in wounded leaves (Yan et al., 2007; Acosta & Farmer, 2010). The authors showed that this induction was correlated with the accumulation of JA and JA-Ile and moreover that it was dependent on COI1. Although most of JAZ proteins were shown to be rapidly degraded upon JA-Ile perception, some studies also showed a relative stability of some JAZ proteins following fluctuating JA-Ile levels (Chung et al., 2010). This negative feedback loop probably acts to restrain the duration and amplitude of JA-triggered responses. However, JA is also involved in a so-called positive feedback loop where it can stimulate its own synthesis by inducing MYC2-dependent expression of genes like LOX2, OPR3, AOS and AOC (Browse, 2009). The wound-induced expression of AOS and OPR3 was shown to be COI1-dependent (Devoto et al., 2005).

JA crosstalk with other stress related phytohormones

Plant pathogens are usually divided into biotrophs and necrotrophs based on their lifestyles. Biotrophic pathogens feed on living tissue whereas necrotrophs kill the host tissue and feed on dead residues. While it is generally admitted that JA has a role in defense against necrotrophic pathogens and herbivorous insects, SA rather activates resistance against biotrophic pathogens (Thomma et al., 1998). At first glance, SA and JA have antagonistic roles in plants as mutants impaired in one of them display enhanced expression of marker genes of the other (Kloek et al., 2001; Glazebrook, 2005). However, the reality is more complicated. For instance, the hemi-biotrophic bacteria Pseudomonas syringae induces both JA and SA signaling pathways in tomato (Stout et al., 1999).
NPR1 was described as the receptor of SA. When SA levels increase, NPR1, which is present as a homodimer, is “monomerized” and goes to the nucleus where it can regulate the expression of specific genes. The JA/SA antagonism principally occurs at the transcriptional level where SA can suppress the induction of JA-dependent genes (Spoel et al., 2003; Caarls et al., 2015). The well-known JA marker gene PDF1.2 is under the control of several transcription factors among which ORA59 from the ERF family (Ethylene Responsive Factor) that is activated by ethylene (ET) and JA. ORA59 was shown to be degraded by SA thus blocking the expression of PDF1.2. This suppression of PDF1.2 expression still occurs in coi1-1 mutants showing that the SA/JA crosstalk is independent on COI1 and is likely to occur downstream. Moreover, redox signaling was shown to be important for SA-induced suppression of JA responses. High SA levels lead to a reduced redox state. Inhibition of glutathione synthesis, a reducing agent, blocked the SA-mediated antagonism of PDF1.2 expression. However, the results of JA and SA crosstalk can vary with hormone concentrations and the type of stimuli (Mur et al., 2006; Spoel et al., 2007).

Table of contents :

1. Sensing cell wall integrity and cell death
2. Ion fluxes and other second messengers
3. Production of Reactive Oxygen Species (ROS)
4. Damage-Associated Molecular Patterns (DAMPs) are intercellular signaling molecules produced upon wounding
5. JA is a central actor of plant responses to wounding and insects
6. Long-distance signaling
7. How to differentiate herbivory from mechanical wounding?
1. A great transcriptional reprogramming largely dependent on JA
2. Various defense responses
1. MAPKs are important stress responsive modules in plants
2. Spare data suggest a complex MAPK role in wounding and herbivory interaction
1. MAPKs and signal transduction before this work, a state of knowledge
2. Objectives: characterizing the activation of a MAPK module upon wounding
1. Identification of MAP3Ks able to interact with MKK3 using the yeast 2-hybrid
2. Functional validation of MAP3Ks using a transient expression system
3. Sub-clade III MAP3Ks are strongly transcriptionally regulated by stresses
4. Discussion
1. Wounding activates an MKK3 module in Arabidopsis thaliana
2. The wounding-induced activation of the iconic MPK3 and 6 is not dependent on MKK3
3. The hunt of upstream MAP3K(s)
4. Discussion
Characterization of a MAPK module involved in Arabidopsis response to wounding
1. Exogenous application of some candidate second messengers can activate an MKK3-dependent module
2. Wounding-induced activation of the MKK3-dependent module is disrupted in mutants of the JA signaling pathway
3. MKK3-MPK2 is not activated in systemic leaves
4. Discussion
1. Botrytis cinerea activates an MKK3-dependent module in Arabidopsis thaliana 68
2. Spodoptera littoralis activates an MKK3-dependent module in Arabidopsis thaliana
3. Discussion
1. Plants impaired in MKK3 signaling do not show clear detectable phenotype upon Botrytis infection
2. Plants impaired in MKK3 signaling do not properly respond to herbivorous insects
3. Wounding-induced trichome formation
4. Transcriptomic analysis of mkk3-1 plants submitted to wounding did not highlight any MKK3-regulated genes
5. Discussion
1. Toward a sequential and interdependent activation of two MAPK modules
2. Spatial organization of MAPK activation upon wounding
1. An emerging general working model…
2. …coexisting with other models?
3. Open questions about the functioning of sub-clade III MAP3Ks-MKK3-C-group MAPKs modules
1. A well-conserved MAPK through evolution
2. A role in herbivory signaling?
3. Characterized phenotypes in other stress and developmental contexts
4. The hunt of MPK1/2/7/14 substrates
1. Plant material
2. Culture media and conditions
3. Antibodies
4. Vector backbones
5. Buffers and solutions
1. Plant methods
2. Molecular biology methods
3. Biochemistry methods
ANNEX: Review – Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules


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